Measles virus mutant antigen

ABSTRACT

Disclosed is a measles virus mutant antigen consisting essentially of a measles virus mutant H protein antigen, wherein said measles virus mutant H protein antigen is at least one member selected from the group consisting of the following amino acid sequences (a) to (c): (a) an amino acid sequence of SEQ ID NO: 10; (b) an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; and (c) an amino acid sequence of SEQ ID NO. 4 or SEQ ID NO: 12 By the use of the measles virus mutant antigen of the present invention, it has become possible to provide efficiently and economically a live attenuated measles vaccine which is adapted for an epidemic strain of measles virus, and a diagnostic reagent capable of accurately detecting infections with an epidemic strain of measles virus.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/JP98/02481 which has an Internationalfiling date of Jun. 4, 1998 which designated the United States ofAmerica.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a measles virus mutant antigen and agene coding for the same. More particularly, the present invention isconcerned with a measles virus mutant antigen comprising at least oneprotein antigen selected from the group consisting of a measles virusmutant H protein antigen and a measles virus mutant F protein antigen,and a measles virus mutant gene coding for the measles virus mutantantigen. By the use of the measles virus mutant antigen or the genecoding for the same of the present invention, it has become possible toprovide efficiently and economically a live attenuated measles vaccine.or gene vaccine which is adapted for an epidemic strain of measlesvirus, and a diagnostic reagent capable of accurately detectinginfections with an epidemic strain of measles virus.

2. Prior Art

(1) Pathogenicity: Measles virus is the pathogen of measles, and it isdistributed widely throughout the world. This virus is highlyinfectious, and when a person suffers a droplet infection with a measlesvirus, damage occurs mainly in the respiratory system andreticuloendothelial tissue, thereby causing an acute disease. A personsuffering from measles shows systemic symptoms, such as high fever,catarrh and rash. Further, in severe cases, measles is complicated withbacterial pneumonia, tympanitis and acute encephalitis. In 1996, thenumber of measles patients and number of deaths due to measles in theworld were estimated to be about 42 million and about 1 million 10thousand, respectively [“The World Health Report 1997”, p. 15, WHO(World Health Organization) published in 1997]. As apparent from theabove, measles is an infectious disease which should be taken intoserious consideration, and eradication of measles by vaccines is desiredthroughout the world. In this situation, the Expanded Program onImmunization (EPI) of World Health Organization (WHO) has alreadyadopted a measles eradication program with the goal of controllingmeasles by the year 2010.

(2) Viral morphology and genomic structure: According to the SixthReport of the International Committee on Taxonomy of Viruses, themeasles virus is classified under the order Mononegavirales, familyParamyxoviridae, genus Morbillivirus. The virion of the measles virus isgenerally spherical (diameter: about 150 nm) and has an envelopecomposed of a lipid bilayer. On the surface of the envelope arespike-like projections composed of an H (hemagglutinin) protein andcomposed of an F (fusion) protein, and the bases of the projections(proteins) are supported by a matrix membrane protein at the inner layerof the envelope. The nucleocapsid present in the inside of the envelopeconsists of measles virus genomic RNA which is a linear, non-segmented(−) sense (that is, mononega) RNA having a length of about 16 kb, andproteins. The genomic RNA codes for N (nucleocapsid-associatedproteins), P/C/V (phosphoprotein/C protein/V protein: coded for bytricistronic gene), M (matrix protein), F (fusion protein), H(hemagglutinin protein) and L (large putative polymerase protein), andthe coding regions are located in this order from the 3′ end to the 5′end of the genome (“Virus Taxonomy: Sixth Report of the InternationalCommittee on Taxonomy of Viruses”, Archives of Virology, Supplement 10,pp. 268-270 and pp. 271-272, 1995).

(3) Conventional virus strain for live attenuated measles vaccine:Examples of the virus strains known for live attenuated measles vaccineare: CAM-70, Schwarz FF8, AIK-C, AIK-HDC, TD97, Moraten, Connaught,Schwarz, Edmonston B, Edmonston-Zagreb, Leningrad-16, Shanghai-191,Changchum-47 and Beijing (S. A. Plotokin and E. A. Mortimer, “Vaccines”,2nd edition, pp. 238-239, published by W. B. Saunders Company, 1994).These virus strains are either a host-range mutant or a temperaturemutant of measles virus which are attenuated to ensure safety andeffectiveness so as to be used as an active component for a livevaccine, and such viruses are obtained by sequentially subjecting anisolated strain (wild measles virus) to passages of culture underdifferent conditions prepared by combining various factors, such as hostcell, culture temperature, and pH and composition of a culture medium.

(4) Prevention: Vaccines for preventing measles were put to practicaluse in the early 1960's. At the beginning, the majority of the measlesvaccines used was killed (or inactivated) vaccines (abbreviated “K”)containing killed measles viruses as an active component of the vaccine.However, the killed measles vaccine had an unsatisfactory immunologicaleffect, and further, it induced serious atypical measles. In thissituation, the use of a live vaccine (abbreviated “L”) containing liveattenuated measles viruses as an active component of the vaccinegradually became predominant in the late 1960's. A combination of K andL vaccines was adopted, but since the 1970's, a further attenuated livevaccine (abbreviated “FL”) obtained by further attenuating theabove-mentioned live vaccine virus has become commercially availablethroughout the world for practical use. With respect to the livevaccine, each of the live attenuated measles vaccine strains mentionedin item (3) above is used as an active component of the vaccine.

(5) Problems of measles vaccine and diagnosis: With respect to themaintenance of immunity obtained by using a conventional live attenuatedmeasles virus vaccine, some problems have arisen since the early 1970's.Illustratively stated, reports on secondary vaccine failure and modifiedmeasles have been made, in which it is reported that, people who havebeen vaccinated with measles vaccine were reinfected with measles andsuffered from symptoms which are different from that of the naturalinfection (in general, the symptoms are mild compared to those of thenatural infection, but serious in rare cases). Such reports onreinfection in various parts of the world were made sporadically in thelatter half of the 1980's, and the reports are frequently made in the1990's. Therefore, the development of means for preventing thereinfection and for determining the infecting virus has been earnestlydesired by not only the people in various countries of the world, butalso by the WHO from the viewpoint of the above-mentioned eradicationprogram on measles. However, a measles vaccine or diagnostic reagenteffective for preventing the infection with the currently prevailingmeasles viruses has not yet been realized.

SUMMARY OF THE INVENTION

The inventors of the present invention have not only studied measlesfrom the viewpoint of clinics, epidemiology and vaccine, but alsostudied various measles viruses, such as vaccine strains, epidemicstrains and isolated fresh strains, from the viewpoint of virology andimmunology, together with the genetic analyses of these virus strains.In particular, the primary inventor of the present invention has beencontinuing his studies for more than 30 years. The inventors of thepresent invention have further made extensive and intensive studies forelucidating the differences in antigenicity or immunogenicity betweenconventional virulent strains, and virulent mutants including epidemicstrains, and also for identifying the causes of such differences. As aresult, they have surprisingly found that, with respect to the mutants,the specific regions in each of the genes coding for the H protein and Fprotein possess mutations which result in amino acid substitutions.Further, the inventors of the present invention have found that themutated regions in the H protein and F protein are effective as mutantantigens of the measles virus. The present invention has been completed,based on these novel findings.

Therefore, it is an object of the present invention to provide a measlesvirus mutant antigen, comprising at least one protein antigen selectedfrom the group consisting of a measles virus mutant H protein antigenand a measles virus mutant F protein antigen, which is advantageous forpreparing a vaccine and a diagnostic reagent for a virus of epidemicmeasles.

It is a further object of the present invention to provide a measlesvirus mutant gene, comprising at least one gene selected from the groupconsisting of a gene coding for a measles virus mutant H protein antigenand a gene coding for a measles virus mutant F protein antigen, which isadvantageous for preparing a gene vaccine and a diagnostic reagent for avirus of epidemic measles.

The foregoing and other objects, features and advantages of the presentinvention will be apparent to those skilled in the art from thefollowing detailed description and the appended claims taken inconnection with the accompanying sequence listing.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

In each of SEQ ID NOs: 1 to 20, the left end and the right end of theamino acid sequence are the N-terminus and the C-terminus, respectively.

SEQ ID NO: 1 is the nucleotide sequence of the cDNA corresponding to thegenomic RNA coding for the H protein of the attenuated measles virusCAM-70 strain and the whole amino acid sequence encoded by thenucleotide sequence;

SEQ ID NO: 2 is the whole amino acid sequence of the H protein of theattenuated measles virus CAM-70 strain;

SEQ ID NO: 3 is the amino acid sequence of the fragmentary peptideconsisting of the 93rd to 616th amino acids in SEQ ID NO: 2;

SEQ ID NO: 4 is the amino acid sequence of the fragmentary peptideconsisting of the 176th to 316th amino acids in SEQ ID NO: 2;

SEQ ID NO: 5 is the amino acid sequence of the fragmentary peptideconsisting of the 172nd to 178th amino acids in SEQ ID NO: 2;

SEQ ID NO: 6 is the amino acid sequence of the fragmentary peptideconsisting of the 238th to 244th amino acids in SEQ ID NO: 2;

SEQ ID NO: 7 is the amino acid sequence of the fragmentary peptideconsisting of the 277th to 282nd amino acids in SEQ ID NO: 2;

SEQ ID NO: 8 is the amino acid sequence of the fragmentary peptideconsisting of the 301st to 307th amino acids in SEQ ID NO: 2;

SEQ ID NO: 9 is the nucleotide sequence of the cDNA corresponding to thegenomic RNA coding for the H protein of the virulent measles virus NAstrain and the whole amino acid sequence encoded by the nucleotidesequence;

SEQ ID NO: 10 is the whole amino acid sequence of the H protein of thevirulent measles virus NA strain;

SEQ ID NO: 11 is the amino acid sequence of the fragmentary peptideconsisting of the 93rd to 616th amino acids in SEQ ID NO: 10;

SEQ ID NO: 12 is the amino acid sequence of the fragmentary peptideconsisting of the 176th to 316th amino acids in SEQ ID NO: 10;

SEQ ID NO: 13 is the amino acid sequence of the fragmentary peptideconsisting of the 172nd to 178th amino acids in SEQ ID NO: 10;

SEQ ID NO: 14 is the amino acid sequence of the fragmentary peptideconsisting of the 238th to 244th amino acids in SEQ ID NO: 10;

SEQ ID NO: 15 is the amino acid sequence of the fragmentary peptideconsisting of the 277th to 282nd amino acids in SEQ ID NO: 10;

SEQ ID NO: 16 is the amino acid sequence of the fragmentary peptideconsisting of the 301st to 307th amino acids in SEQ ID NO: 10;

SEQ ID NO: 17 is the nucleotide sequence of the cDNA corresponding tothe genomic RNA coding for the F protein of the attenuated measles virusCAM-70 strain and the whole amino acid sequence encoded by thenucleotide sequence;

SEQ ID NO: 18 is the whole amino acid sequence of the F protein of theattenuated measles virus CAM-70 strain;

SEQ ID NO: 19 is the nucleotide sequence of the cDNA corresponding tothe genomic RNA coding for the F protein of the virulent measles virusNA strain and the whole amino acid sequence encoded by the nucleotidesequence; and

SEQ ID NO: 20 is the whole amino acid sequence of the F protein of thevirulent measles virus NA strain.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, there is provided a measlesvirus mutant antigen, comprising at least one protein antigen selectedfrom the group consisting of (I) a measles virus mutant H proteinantigen and (II) a measles virus mutant F protein antigen,

the measles virus mutant H protein antigen (I) being at least one memberselected from the group consisting of the following amino acid sequences(a) to (f) identified with the positional amino acid numbers of eitherSEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238thto 244th amino acids, the 277th to 282nd amino acids, and the 301st to307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 174th,176th, 243rd, 279th and 302nd amino acids, and neighboring amino acidsof the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10,wherein the fragmentary, contiguous sequences are exclusive of thefragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 93rd,157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th,338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th,603rd and 616th amino acids, and neighboring amino acids of the selectedamino acid in either SEQ ID NO: 2 or SEQ ID NO: 10 wherein thefragmentary, contiguous sequences are exclusive of the fragmentarysequences (d) and (e); and

the measles virus mutant F protein antigen (II) being at least onemember selected from the group consisting of the following amino acidsequences (g) and (h) identified with the positional amino acid numbersof either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 11th,52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboringamino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ IDNO: 20.

For easy understanding of the present invention, the essential featuresand various preferred embodiments of the present invention areenumerated below.

1. A measles virus mutant antigen, comprising at least one proteinantigen selected from the group consisting of (I) a measles virus mutantH protein antigen and (II) a measles virus mutant F protein antigen,

the measles virus mutant H protein antigen (I) being at least one memberselected from the group consisting of the following amino acid sequences(a) to (f) identified with the positional amino acid numbers of eitherSEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238thto 244th amino acids, the 277th to 282nd amino acids, and the 301st to307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 174th,176th, 243rd, 279th and 302nd amino acids, and neighboring amino acidsof the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10,wherein the fragmentary, contiguous sequences are exclusive of thefragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 93rd,157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th,338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th,603rd and 616th amino acids, and neighboring amino acids of the selectedamino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein thefragmentary, contiguous sequences are exclusive of the fragmentarysequences (d) and (e); and

the measles virus mutant F protein antigen (II) being at least onemember selected from the group consisting of the following amino acidsequences (g) and (h) identified with the positional amino acid numbersof either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 11th,52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboringamino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ IDNO: 20.

2. A measles virus mutant gene, comprising at least one gene selectedfrom the group consisting of (I) a gene coding for a measles virusmutant H protein antigen and (II) a gene coding for a measles virusmutant F protein antigen,

the gene coding for a measles virus mutant H protein antigen (I) beingat least one member selected from the group consisting of the followinggenes (a) to (f) identified with the positional amino acid numbers ofeither SEQ ID NO: 2 or SEQ ID NO: 10:

(a) a gene coding for the whole sequence of the 1st to 617th aminoacids;

(b) a gene coding for a fragmentary sequence of the 93rd to 616th aminoacids;

(c) a gene coding for a fragmentary sequence of the 176th to 316th aminoacids;

(d) genes coding for fragmentary sequences of the 172nd to 178th aminoacids, the 238th to 244th amino acids, the 277th to 282nd amino acids,and the 301st to 307th amino acids;

(e) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein thesequences each comprise an amino acid selected from the group consistingof the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboringamino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ IDNO: 10, wherein the genes are exclusive of the genes (d); and

(f) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein thesequences each comprise an amino acid selected from the group consistingof the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th,296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th,592nd, 600th, 603rd and 616th amino acids, and neighboring amino acidsof the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10,wherein the genes are exclusive of the genes (d) and (e); and

the gene coding for measles virus mutant F protein antigen (II) being atleast one member selected from the group consisting of the followinggenes (g) and (h) identified with the positional amino acid numbers ofeither SEQ ID NO: 18 or SEQ ID NO: 20:

(g) a gene coding for the whole sequence of the 1st to 550th aminoacids; and

(h) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein thesequences each comprise an amino acid selected from the group consistingof the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, andneighboring amino acids of the selected amino acid in either SEQ ID NO:18 or SEQ ID NO: 20.

Hereinbelow, the present invention is described in detail.

In the present invention, with respect to the nucleotide sequences, Arepresents adenine, C represents cytosine, G represents guanine and Trepresents thymine.

In the present invention, with respect to the amino acid sequences, Alarepresents an alanine residue, Arg represents an arginine residue, Asnrepresents an asparagine residue, Asp represents an aspartic acidresidue, Cys represents a cysteine residue, Gln represents a glutamineresidue, Glu represents a glutamic acid residue, Gly represents aglycine residue, His represents a histidine residue, Ile represents anisoleucine residue, Leu represents a leucine residue, Lys represents alysine residue, Met represents a methionine residue, Phe represents aphenylalanine residue, Pro represents a proline residue, Ser representsa serine residue, Thr represents a threonine residue, Trp represents atryptophan residue, Tyr represents a tyrosine residue and Val representsa valine residue.

For making more clear the essential features of the present invention,the technical features of the present invention will be described indetail below by explaining how the present invention has been developed.

All of the conventional live measles vaccines are produced from virusstrains which were obtained by attenuating the viruses which prevailedin the 1950's and 1960's. Therefore, the antigenicity of conventionalvaccine strains corresponds to the antigenicity of virus strains whichwere epidemic half a century ago.

On the other hand, it has been found that the most recent epidemicstrains and the relatively recent epidemic strains have mutations in theH protein gene and the F protein gene which are genes responsible for avirion to adsorb on and penetrate into cells to thereby cause aninfection with the virus. Specifically, the mutation in the H proteingene causes substitution of 17 to 19 amino acids in a specific region inthe whole amino acid sequence (consisting of 617 amino acids) of the Hprotein and, such a substitution changes the three-dimensional structureof the protein, so that an antigenic mutation occurs. This antigenicmutation is as large as the antigenic shift of the H protein, andimportant.

Further, the present inventors have found that the antigenic mutation ofthe epidemic strain is an important factor causing the above-mentionedsecondary vaccine failure and modified measles.

Based on these findings, the present inventors have succeeded inproviding a viral genome of a measles virus mutant, particularly amutant H protein gene and a mutant F protein gene, and the mutantantigens (not only the whole protein but also fragmentary peptidesthereof) encoded by the genes.

In addition, the present inventors have successfully developed thefollowing utilities (i) to (iii) of the above-mentioned genes, mutantantigens and their epitopes, and the like.

(i) Modification of a viral genome of a live vaccine strain: Arecombinant virus is prepared by replacing the H protein gene of aconventional live vaccine strain with the H protein gene of an epidemicstrain. By using this method, a live attenuated vaccine strain which isadapted for the antigenicity of the epidemic strain is obtainedspeedily. In other words, the recombinant virus obtained in theabove-mentioned manner can be used as an active component of anexcellent vaccine which is capable of effectively preventing infectionswith the epidemic strains. This a method is also advantageous from aneconomical view-point. That is, the time, labor and costs necessary forattenuating a virus can be decreased to a large extent. As mentionedabove, with respect to the production of conventional vaccines, there isno specific limitation on the method for attenuating viruses, andconventionally, the attenuation was conducted mainly by passage, whichrequires at least several years to about 10 years for establishing anattenuated strain for a live vaccine.

(ii) Preparation of an active component for a gene vaccine: A genevaccine is prepared by inserting the H protein gene and the F proteingene of an epidemic strain into various vectors, such as a plasmidvector, a cosmid vector, a phage vector, a shuttle vector, a viralvector of a non-proliferating viral vector and the like.

For example, when a non-proliferating recombinant virus, which isprepared by inserting the cDNAs for the above-mentioned H protein geneand F protein gene into a non-proliferating viral vector, is used as anactive component for a gene vaccine or DNA vaccine, such a vaccine iscapable of inducing both humoral immunity and cellular immunity like aconventional live measles vaccine. A remarkable feature of this vaccineis that nasal injection is possible.

In addition, a cDNA fragment comprising the mutated region of the Hprotein gene of an epidemic strain can be inserted into, for example, aplasmid vector, to prepare a naked DNA. The thus prepared naked DNA canalso be used as an active component for a DNA vaccine or gene vaccinefor preventing measles.

(iii) Preparation of a suitable reagent for diagnosis of epidemicstrains: PCR primers are synthesize so that the synthesized primersreflect the mutations in the H protein gene or F protein gene of theepidemic strains. The synthesized primers can be used as a reagent forgene diagnosis not only for identifying the epidemic strains, but alsofor differentiating a virulent strain from an attenuated strain, or viceversa.

Further, the mutant antigens (whole proteins or fragmentary peptidesthereof) encoded by the above-mentioned genes are prepared, and theirepitopes are chemically synthesized. The antigens and epitopes areprovided as suitable antigens for diagnosis of epidemic measles.

An explanation is made below with respect to the preparation of ameasles virus mutant antigen and a measles virus mutant gene of thepresent invention, and the use of the prepared antigens and genes as avaccine and a diagnostic reagent.

I Preparation of Measles Virus Mutant Antigen and Measles Virus MutantGene

(1) Antigen analysis of various measles virus antigens: The antigenicityof the measles virus mutant antigen can be evaluated by a neutralizationtest, an HI (hemagglutination inhibition) test, a PA (passiveagglutination) test, an enzyme immunoassay and a fluorescent antibodytechnique each using a monoclonal antibody, and the like. However, fordetermining the effectiveness of the virus antigen as antigen for avaccine, it is requisite to evaluate the antibody titer by theneutralization test, and it can be performed in accordance with themodified Ueda method (Biken Journal, 14, 155-160, 1971) which employsmicroplates.

With respect to the antibodies used in the antigen analysis, sera, suchas a serum from a measles patient and mouse immune sera against measlesviruses as mentioned below, can be employed.

With respect to the antigens (challenge viruses) used in the antigenanalysis, it is important to select different measles strains from thestrains isolated in the past to the present. Representative examples ofepidemic strains of the 1950's and 1960's (virulent strains of the past)include Tanabe strain and Edmonston strain; and examples of live vaccinestrains established by attenuating the above-mentioned virulent strains(conventional attenuated strains) include CAM-70 strain and Edmonston Bstrain. As the recent epidemic strains (virulent strains), use can bemade of the measles strains isolated in various countries in the 1990's.For example, the virus strains isolated from various resources by thepresent inventors, such as F-t strain (isolated in 1991 from throat swabof a reinfected patient), F-b strain (isolated in 1991 from blood of areinfected patient), U-t strain (isolated in 1991 from throat swab of anon-vaccinated patient), U-b strain (isolated in 1991 from blood of anon-vaccinated patient), Momo strain (isolated in 1995 from a patient)and NA strain (isolated in 1996 from a patient) can be used as therecent epidemic strain.

Hereinafter, the following strains will be frequently referred to asindicated in the parentheses: Tanabe (Tana) strain, Edmonston (Edmo)strain, CAM-70 (CAM) strain and Momo (MO) strain.

(2) Determination of the mutated regions in the nucleotide sequence of agene, and translation of the gene into an amino acid sequence: Theanalysis of the viral genome of each of the measles strains mentioned initem (1) above is carried out as follows. First, the viral RNA genome isextracted and the cDNA is prepared using primers. The nucleotidesequence of the prepared cDNA is determined by the direct sequencingmethod which employs PCR method (hereinafter, simply referred to as“PCR-direct sequencing method”). The search for DNA sequence homologybetween different measles virus strains is performed while determiningthe nucleotide sequence of the genes, to thereby specify the mutatedregions within the genes.

Next, each of the above-specified mutated regions are translated intoamino acid sequence in accordance with the universal code, and thedeductive analyses of the amino acid sequences are performed as follows.Analysis of the hydrophobicity pattern and determination of thesecondary structure of a protein by Chou-Fasman analysis are performedby computer using the computer software “DNASIS-Mac (version 3.6)”(manufactured and sold by Hitachi Software Engineering Co., Ltd.,Japan). Epitopes can be identified, for example, by computer using thecomputer software “Epitope Advisor” [manufactured and sold by FujitsuKyushu System Engineering (FQS) Ltd., Japan].

(3) Measles virus mutant antigens and genes coding for the same: Basedon the antigen analyses mentioned in item (1) above-and the studies onthe nucleotide and amino acid sequences mentioned in item (2) above, thepresent inventors have conducted comparative analyses between thestrains of recent epidemic measles, the virulent strains of the past andthe conventional strains for a live attenuated measles vaccine, and theyidentified the respective regions in the H protein and the F proteinwhich contain amino acid substitutions. Further, the present inventorsspecified the antigens useful for the vaccine or the reagent fordiagnosis of epidemic strain of measles virus. The measles virus mutantantigen of the present invention is the whole protein or a fragmentarypeptide of the H protein and F protein of the attenuated measles virusCAM-70 strain or the epidemic measles virus NA strain. Each of the aminoacid sequences is disclosed for the first time by the inventors of thepresent invention. Specifically, the measles virus mutant antigen of thepresent invention is an antigen comprising at least one protein antigenselected from the group consisting of (I) an H protein antigen of ameasles mutant and (II) an F protein antigen of a measles mutant.

The measles virus mutant H protein antigen (I) is at least one memberselected from the group consisting of the following amino acid-sequences(a) to (f) identified with the positional amino acid numbers of eitherSEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238thto 244th amino acids, the 277th to 282nd amino acids, and the 301st to307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 174th,176th, 243rd, 279th and 302nd amino acids, and neighboring amino acidsof the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10,wherein the fragmentary, contiguous sequences are exclusive of thefragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 93rd,157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th,338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th,603rd and 616th amino acids, and neighboring amino acids of the selectedamino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein thefragmentary, contiguous sequences are exclusive of the fragmentarysequences (d) and (e).

The measles virus mutant F protein antigen (II) is at least one memberselected from the group consisting of the following amino acid sequences(g) and (h) identified with the positional amino acid numbers of eitherSEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids ineither SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences eachcomprise an amino acid selected from the group consisting of the 11th,52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboringamino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ IDNO: 20.

Among the protein antigens included in the measles virus mutant antigensof the present invention, the protein antigens as defined in items (a)and (g) above are H protein and F protein, respectively, and the proteinantigens as defined in items (b) to (f) and (h) above are peptides(fragmentary sequences). Further, the four fragmentary sequences asdefined in item (d) above, namely, the fragmentary sequences of the172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to282nd amino acids, and the 301st to 307th amino acids, identified withthe positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO:10, are epitopes of the H protein which are disclosed for the first timeby the inventors of the present invention. With respect to the proteinantigens as defined in items (a) to (d) and (g) above, the specificsequences are shown in the Sequence Listing. Each of the antigens of thepresent invention can be chemically synthesized, based on the sequencesshown in the Sequence Listing (see Example 5).

The measles virus mutant antigen of the present invention comprises atleast one protein antigen selected from the group consisting of theabove-mentioned whole proteins and fragmentary peptides, and the proteinantigen can be chosen, based on the intended utility of the measlesvirus mutant antigen. Occasionally, several protein antigens can be usedin combination.

In a further aspect of-the present invention, a gene coding for theabove-mentioned measles virus mutant antigen is provided. Specifically,the measles virus mutant gene comprising at least one gene selected fromthe group consisting of (I) a gene coding for an H protein antigen of ameasles mutant and (II) a gene coding for an F protein antigen of ameasles mutant is provided.

The gene (I) coding for a measles virus mutant H protein antigen is atleast one member selected from the group consisting of the followinggenes (a) to (f) identified with the positional amino acid numbers ofeither SEQ ID NO: 2 or SEQ ID NO: 10:

(a) a gene coding for the whole sequence of the 1st to 617th aminoacids;

(b) a gene coding for a fragmentary sequence of the 93rd to 616th aminoacids;

(c) a gene coding for a fragmentary sequence of the 176th to 316th aminoacids;

(d) genes coding for fragmentary sequences of the 172nd to 178th aminoacids, the 238th to 244th amino acids, the 277th to 282nd amino acids,and the 301st to 307th amino acids;

(e) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein saidsequences each comprise an amino acid selected from the group consistingof the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboringamino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ IDNO: 10, wherein the genes are exclusive of the genes (d); and

(f) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein saidsequences each comprise an amino acid selected from the group consistingof the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th,296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th,592nd, 600th, 603rd and 616th amino acids, and neighboring amino acidsof the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10,wherein the genes are exclusive of the genes (d) and (e).

The gene (II) coding for measles virus mutant F protein antigen is atleast one member selected from the group consisting of the followinggenes (g) and (h) identified with the positional amino acid numbers ofeither SEQ ID NO: 18 or SEQ ID NO: 20:

(g) a gene coding for the whole sequence of the 1st to 550th aminoacids; and

(h) genes coding for fragmentary, contiguous sequences of at least 6amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein thesequences each comprise an amino acid selected from the group consistingof the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, andneighboring amino acids of the selected amino acid in either SEQ ID NO:18 or SEQ ID NO: 20.

With respect to the gene coding for the measles virus mutant antigen ofthe present invention, there is no particular limitation as long as thegene codes for the whole protein or a fragmentary peptide of the measlesvirus mutant antigen. Therefore, the gene is not limited to thenucleotide sequence of the genomic RNA of CAM-70 strain or NA strain. Asthe measles virus mutant gene, use can be made of the cDNAs shown in SEQID NOs: 1, 9, 17 and 19, or the gene can be prepared by synthesizing anucleotide sequence on the basis of an amino acid sequence of a measlesvirus mutant antigen.

The measles virus mutant gene of the present invention comprises atleast one gene selected from the group consisting of the above-mentionedgenes, and the gene can be chosen, based on the intended utility of themeasles virus mutant gene. Like the measles virus mutant antigen of thepresent invention, the measles virus mutant gene of the presentinvention comprises both the genes of the attenuated strain and thegenes of the epidemic strain. Based on the disclosure of the presentinvention, for example, a live vaccine effective for preventing theinfection with the epidemic strains can be produced {see thebelow-mentioned item [II](1), and Examples 2 and 3}. When several genesare used in combination, they can also be used in such a form as ligatedto each other {see the below-mentioned item [II](2) and Example 4}.

The antigens and genes coding for the same of the present invention,which respectively comprise the above-mentioned sequences, are effectiveas a marker for identifying a virulent strain or an attenuated strain,and are also important and advantageous for improving conventionalvaccines and developing diagnostic reagents.

II Use of Measles Virus Mutant Antigen and Measles Virus Mutant Gene ofthe Present Invention as Vaccine and Diagnostic Reagent

(1) Preparation of an effective live vaccine for epidemic measlesstrains: A recombinant virus is prepared by replacing a gene of a livevaccine strain with a corresponding gene of an epidemic strain. Withrespect to the live vaccine strain, various strains mentioned under“Prior Art” of the specification can be used, but preferably, use ismade of a strain which has been employed as an active component of alive vaccine in various countries for at least 10 years. That is, astrain having approved safety and effectiveness as an active componentfor a vaccine, such as CAM-70 strain, is preferred.

With respect to the epidemic strain used for preparing a live vaccine,the epidemic strain is selected so that when the selected strain iscompared with a live vaccine strain, the epidemic strain has a marked,broad antigenic mutation due to the genetic mutation thereof.Specifically, a preferred epidemic strain is a recent epidemic strainwhich is being isolated at high frequency and is widely prevailing, andwhich has a universal antigenic mutation (that is, an antigenic mutationwhich is not peculiar to a particular strain), for example, MO strain orNA strain isolated by the inventors of the present invention in 1995 to1996.

With respect to the gene to be used for replacement, the gene can beselected from the genes coding for the antigens mentioned in item [I](3)above, and the genes can be used individually or in combination.However, for preventing any possible reversion of a recombinant virusfrom an attenuated state to a virulent state, and also from theviewpoint of ease in preparing a recombinant virus, it is preferred touse the gene described in item (c), that is, the gene coding for thefragmentary sequence of the 176th to 316th amino acids of the H protein(the peptide of SEQ ID NO: 4 and the peptide of SEQ ID NO: 12). Theamino acid substitutions in this region are characteristic of theepidemic strains and such substitutions are not found in the H proteinof the attenuated strains. Therefore, by using the gene coding for thisregion, an epitope of the epidemic strain can be introduced into theattenuated strain without changing the sequences which arecharacteristic of the attenuated strains (that is, the sequences whichmay be causative of attenuation).

The recombinant virus can be produced by the method of Radecke et al.(EMBO Journal, Vol. 14, No. 23, pp. 5773-5784, 1995) which is a methodfor genetic recombination of a non-segmented negative-strand RNA viral(mononegaviral) genome, or by the modified method of Radecke et al.,which has been developed by the inventors of the present invention.

The method of Radecke et al. (frequently referred to as “reversegenetics”) will be explained below. First, the cells of 293 cell line(American Type Culture Collection, Accession No. ATCC CRL-1573) weretransfected with a recombinant vector containing genes coding for T7 RNApolymerase and measles virus N protein and P protein, thereby obtainingtransfectants (i.e., helper cells) capable of expressing T7 RNApolymerase, N protein and P protein. Next, an expression vector capableof expressing L protein (polymerase) of the measles virus under thecontrol of T7 promoter is constructed (hereinafter, the constructedexpression vector is simply referred to as “V1”). Further, a cDNA forthe (+) sense RNA of the whole genome of CAM-70 strain is prepared, anda DNA fragment coding for a region in the H protein which contains theabove-mentioned amino acid substitutions is cleaved and removed from thecDNA for CAM-70 strain by means of restriction enzymes. Then, the DNAsequence of the corresponding region of the viral genome of epidemic MOstrain or NA strain is prepared therefrom and inserted into therestriction site of the cDNA for CAM-70 strain, to thereby obtain arecombinant cDNA. The obtained recombinant cDNA is inserted into plasmidpBluescript SK or KS (manufactured and sold by Stratagene Co., Ltd.,England), thereby obtaining an expression vector (hereinafter, theobtained expression vector is simply referred to as “V0”), wherein theexpression vector is prepared so that the recombinant cDNA is capable oftranscription by T7 RNA polymerase. V0 and V1 are co-transfected to thehelper cells prepared above, and the desired recombinant measles viruscan be obtained by subsequently culturing the transfected cells. Theproliferation of the recombinant virus in the transfected cells can beconfirmed by detecting the occurrence of CPE (cytopathic effect),wherein the transfected cells generate syncytia, or by conducting amicroscopic observation using a fluorescent antibody technique with amonoclonal antibody against the epitope of the protein encoded by thereplaced gene.

The recombinant attenuated measles virus of CAM-70 strain, in which thegene coding for the 176th to 316th amino acids of the H protein ofCAM-70 strain (SEQ ID NO: 4) is replaced by the gene coding for the176th to 316th amino acids of the H protein of MO strain or NA strain(SEQ ID NO: 12), is obtained by using the above-mentioned method.

Further, the modified method of Radecke et al. is explained below. Thismodified method is such that the helper cells are not required, and anydesired permissive cells can be used as host cells for the recombinantvirus. With respect to the host cells employed, cells which are ensuredto be safe as a culture host for the live vaccine strains and areapproved as the host cells therefor, such as, MRC-5 cells and WI-38cells are preferably used to prevent an introduction of an unidentifiedfactor, a carcinogen and the like into the virus. First, the expressionvectors for each of the genes coding for N, P, and L proteins of CAM-70strain are individually prepared using plasmids pcDNA3.1(+) orpcDNA3.1(−) (manufactured and sold by Invitrogen Co., Ltd., Canada). Forexample, each of the genes encoding N, P and L proteins of CAM-70 strainis individually inserted into an appropriate restriction site ofpcDNA3.1(−), thereby constructing the expression vectors. For theexpression of T7 RNA polymerase, recombinant MVA (hereinafter, simplyreferred to as “recMVA”; FEBS Letter, vol. 371, no. 1, pp. 9-12, 1995)can be used. The above-prepared three expression vectors and theexpression vector V0 mentioned in connection with the method of Radeckeet al. are co-transfected to either the MRC-5 cells or WI-38 cells whichhave already been transfected with recMVA. The desired recombinantattenuated measles virus is obtained by culturing the transfected cellsat about 35° to 38° C. The proliferation of the virus can be confirmedby detecting the occurrence of CPE or by conducting the microscopicobservation using the fluorescent monoclonal antibody techniquementioned above. Further, the antigenicity and immunogenicity of theobtained recombinant virus can be qualified in accordance with theantigen analysis mentioned in item [I](1) above.

(2) Preparation of an active component of a gene vaccine: Thenon-proliferating recombinant adenovirus can be prepared by inserting agene of an epidemic measles virus into a non-proliferating adenoviralgenome. The prepared recombinant adenovirus is effective as an activecomponent of a gene vaccine. For preparing the recombinant virus,COS-TPC method developed by Saito et al. [Cell Technology (SaiboKogaku), vol. 13, no. 8, pp. 757-763, 1994] can be employed. In thismethod, DNA-TPC (viral DNA-Terminal Protein Complex) of the genome ofhuman adenovirus 5, and a cassette cosmid carrying almost all of thewhole genome of the non-proliferating adenovirus (cassette cosmidpAdexl; U.S. Pat. No. 5,700,470) are used. The non-proliferatingadenovirus is derived from human adenovirus 5 and it lacks E1A and E1Bgenes which are essential for viral proliferation, and therefore, thisvirus is incapable of proliferation in cells other than the 293 cellswhich constantly express E1A and E1B genes. Further, this virus lacksgene coding for E3 protein, a protein which antagonizes the recognitionof viral antigens by CTL (cytotoxic T lymphocytes). Due to thiscontrived design of the adenovirus, cellular immunity induced by CTL isexpected to develop even in the presence of this virus.

With respect to a measles virus gene used for preparing the recombinantvirus, the gene can be selected from the genes coding for the antigensmentioned in item [I](3) above, and the genes can be used individuallyor in combination. However, for improving the immunogenicity which isnecessary for providing a protection against the viral infection (thatis, adsorption and penetration of a measles virus to a cell), it ispreferred to use in combination the gene coding for the whole H proteinmentioned in item (a) (SEQ ID NO: 2 or SEQ ID NO: 10) and the genecoding for the whole F protein mentioned in item (g) (SEQ ID NO: 18 orSEQ ID NO: 20).

Specifically, the cDNAs for the above mentioned H protein gene and Fprotein gene (for example, the nucleotide sequences of SEQ ID NO: 9 andSEQ ID NO: 19) are prepared [when the cDNAs are ligated, they areligated in the order of H protein—F protein (HF) or F protein—H protein(FH) in the direction of from the 5′ end to the 31 end], and theprepared cDNAs are inserted into the E1A·E1B deletion site of thecassette cosmid pAdex1, to thereby obtain recombinant cosmid pAdex1/HFor pAdex1/FH. On the other hand, DNA-TPC is extracted from the parentadenovirus strain, and the DNA-TPC is digested with the restrictionenzyme Eco T22I (manufactured and sold by Takara Shuzo Co., Ltd.,Japan), to thereby obtain digestion product DNA-TPC/Eco T22I.Subsequently, pAdex1/HF or pAdex1/FH, and DNA-TPC/Eco T22I areco-transfected to the 293 cells by calcium phosphate method. As a resultof the co-transfection, homologous recombination between the transfectedDNAs occurs in the cells, and a non-proliferating recombinant adenoviruscontaining measles virus H protein gene and F protein gene is obtained.The presence of measles virus H and F proteins in the non-proliferatingrecombinant adenovirus can be confirmed by testing Hela cells infectedwith the obtained adeno-virus by fluorescent antibody technique usingthe monoclonal antibodies against the measles proteins.

(3) Production of a measles vaccine: The live attenuated measles vaccinecan be produced by using the recombinant attenuated measles virusmentioned in item [II](1) above as a seed virus. For example, therecombinant virus is proliferated in permissive cells, such as chickenembryo cells, thereby obtaining a virus suspension. The cells areremoved from the obtained virus suspension by low-speed centrifugation,thereby obtaining a supernatant. Then, the obtained supernatant issubjected to filtration to thereby prepare a bulk vaccine solution. Theprepared bulk vaccine solution is diluted with a medium, such as BMEmedium (Eagle's Basal Medium), so as to obtain a vaccine solutioncomprising the virus in a sufficient antigenic amount, for example, notless than 5,000 TCID₅₀ (Median Tissue Culture Infective Dose) per 0.5 mlof vaccine solution. A stabilizer for stabilizing the virus can be addedto the vaccine solution when the bulk vaccine solution is being diluted.Subsequently, the diluted vaccine solution is dispensed into suitablecontainers, such as 1 to 20 ml volume vials, and then, the containersare sealed hermetically, and the sealed vaccine is provided as a vaccinepreparation. The vaccine preparation can be provided as a liquidpreparation or as a lyophilized preparation obtained by lyophilizing thevaccine after dispensation. Prior to the use of the vaccine preparation,it is requisite to subject the vaccine preparation to various tests oneffectiveness and safety to assure its quality as a vaccine. The testsare conducted in accordance with Pharmaceutical Affairs Law (the Law No.145 established in 1960) and a provision entitled “Dried AttenuatedMeasles Virus Live Vaccine” in the Notification No. 217 of the JapaneseMinistry of Health and Welfare: Seibutsugakuteki Seizai Kijun (MinimumRequirements for Biological Products) established in 1993. With respectto the manner of administration, for example, the vaccine preparation isadministrated by subcutaneous injection in an amount of 0.25 to 0.5 mlper dose.

The non-proliferating recombinant virus mentioned in item [II](2) abovecan be produced in large yield using the 293 cells. The recombinantvirus can be prepared from the liquid culture of 293 cells insubstantially the same manner as mentioned above for preparing thevaccine preparation, so that the final virus content of a liquid orlyophilized preparation is 10⁶ to 10⁸ PFU (plaque-forming unit) per 1 mlof preparation. Such virus preparation can be provided as an activecomponent for a gene vaccine. With respect to the manner ofadministration, the gene vaccine can be administered by subcutaneous,intramascular or nasal injection in an amount of 0.25 to 0.5 ml perdose, and from the viewpoint of ease in injection procedure, nasalinjection is especially preferred.

(4) Preparation of a diagnostic reagent: The antigens mentioned in item[I](3) above (whole protein or fragmentary peptide thereof) can be usedindividually or in combination as an antigen for diagnosis. When usingseveral antigens in combination, the antigens containing differentepitopes are preferably used to broaden the spectrum of reactivity withantibody. The antigen of the present invention can be provided as anantigen to be used in various diagnoses, such as diagnosis usingprecipitation reaction, agglutination reaction, neutralization reaction,fluorescent antibody technique, enzyme immunoassay, andradioimmunoassay. Further, the antigens can be inoculatedintraperitoneally, subcutaneously or intramuscularly to an animal, suchas rabbit, guinea pig and mouse, to prepare an immune serum, antibody orthe like. The thus prepared antibody can be also provided as an antibodyfor detecting antigens in various diagnoses.

The antigen or antibody of the present invention is diluted so as toprepare a diagnostic reagent containing the antigen or antibody in anamount sufficient to cause an antigen-antibody reaction.

Further, the genes coding for the antigen mentioned in item [I](3) abovecan be used individually or in combination, for example, as a probereagent for gene diagnosis and a reagent for identifying the measlesvirus strains. PCR primers can be designed, based on the amino acidsequences of the H protein and F protein of the attenuated strain andepidemic strain (for example, SEQ ID NOs: 2, 10, 18 and 20) and basedthe nucleotide sequences of the genes coding for the same (for example,SEQ ID NOs: 1, 9, 17 and 19) which are disclosed in the presentspecification. Such PCR primers can be provided as a reagent fordiagnosis using the PCR method.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in more detail withreference to the following Examples, but they should not be construed aslimiting the scope of the present invention.

EXAMPLE 1

Antigen analyses and gene analyses are performed as follows, to therebyidentify the differences in nucleotide sequences and amino acidsequences between the past epidemic strains (virulent strains),conventional live vaccine strains (attenuated strains) and recentepidemic strains (virulent strains). In addition, the amino acidsequences of mutant antigens and their epitopes are determined.

(1) Antigen Analyses

Measurement of Neutralizing Antibody Titers (1):

Each of the neutralizing antibody titers for a vaccine strain and anepidemic strain in a test serum is measured separately by the modifiedUeda method which employs microplates. As a challenge virus, CAM-70strain is used as a vaccine strain, and Momo strain is used as anepidemic strain. B95a cells are used for proliferating the viruses. Thetest sera are selected from sample sera individually obtained frominfants who received vaccination with measles CAM-70 strain vaccineduring the period of 1994 to 1996, in which the sera were sampled fromeach infant before the vaccination and 1 to 2 months after thevaccination. Two groups of test sera (i.e., groups A and B) areprepared, so that group A consists of eleven (11) sample sera eachhaving HI (hemagglutination inhibition) antibody titer of 8-fold, andgroup B consists of fourteen (14) sample sera each having HI antibodytiter of 64-fold, both measured using HI antigen of Toyoshima strain(isolated in 1959). Two rows of microplate wells were used fordetermining the neutralizing antibody titer in a test serum.

20 μl of culture medium is dispensed into each well of a microplate, andserial 2-fold dilution of each test serum (20 μl) with the dispensedculture medium is performed. 20 μl of a challenge virus solution (viralinfective dose is already adjusted to 10 TCID₅₀/20 μl) is placed in eachwell and mixed with the diluted test serum, and then, a reaction isallowed to proceed at 37° C. for 1 hour. Subsequently, 100 μl ofcultured B95a cells is added to each reaction mixture in the well, andthe cells are cultured for 1 week. The neutralizing antibody titer ismeasured by detecting the occurrence of CPE (cytopathic effect). Theresults are shown below.

Group A: The relative antibody titer (antibody titer for epidemicstrain/antibody titer for vaccine strain) is less than ½ in nine (9)test sera out of the total of eleven (11) test sera (9/11; 81.8%).Particularly, among the above-mentioned nine (9) test sera, the antibodytiter for the epidemic strain is not detected (that is, the value ofantibody titer is less than 0 as expressed in terms of log₂) in six (6)test sera (6/11; 54.5%), even though the antibody titer for the vaccinestrain is from 2.6 to 3.6 (the values are expressed in terms of log₂) inthese test sera. With respect to the remaining two (2) test sera, therelative antibody titer is 1 (i.e., 1/1).

Group B: With respect to ten (10) test sera out of the total of fourteen(14) test sera (10/14; 71.4%), the relative antibody titer (antibodytiter of epidemic strain/antibody titer of vaccine strain) is so low asto fall within the range of from ½ to ⅛. Each of the remaining four (4)test sera has a relative antibody titer of approximately 1 (i.e., 1/1).Since test sera having a relative antibody titer (antibody titer ofepidemic strain/antibody titer of vaccine strain) of less than ½ arefrequently found among the sera containing antibodies against aconventional measles virus, it is concluded that the recent epidemicstrain has certain mutation in its antigens (that is, both the H and Fproteins) which are related to the neutralization of antibodies andviral infection.

Measurement of Neutralizing Antibody Titers (2):

The neutralizing antibody titers in a mouse immune serum against the Hprotein of NA strain is determined in substantially the same manner asmentioned in measurement (1) above, except that CAM-70 strain, Tanabestrain and NA strain are separately used as a challenging virus, and themouse immune sera prepared in the following manner are used as the testsera.

Ten BALB/C mice (4 weeks old) are individually injected intramuscularlywith 100 μl of a solution of expression vector pcDNA3.1(−)/H capable ofexpressing the H protein of NA strain (hereinafter, simply referred toas “naked DNA”). After 2 weeks from the first injection, as a boosterinjection, the intramuscular injection of the naked DNA is repeated insubstantially the same manner as the first injection. As a control,physiological saline is injected instead of the naked DNA to each of thefive mice in substantially the same manner as mentioned above. After 4weeks from the booster injection, blood is sampled individually fromeach of the ten immunized mice and five control mice to thereby obtainmouse immune sera.

The above-mentioned naked DNA is the expression vector for NA strain Hprotein prepared in connection with Example 3 below, and it is obtainedby amplifying plasmid pcDNA3.1(−)/H in E. coll, and substantiallypurifying the plasmid from the E. coli culture.

As a result, it is found that the average neutralizing antibody titer(the value expressed in terms of log₂) for NA strain, CAM-70 strain andTanabe strain is 4.0, 3.8 and 4.0, respectively. From the above resultsand the results of measurement (1) mentioned above, it is concluded thatthe antigen of the recent epidemic strain (i.e., NA strain) has abroader spectrum with respect to the reactivity with antibody than thatof the vaccine strain or the epidemic strain of the past, and that theantigenicities of the vaccine strain and the past epidemic strain areembraced by that of the recent epidemic strain.

(2) Gene Analyses

Determination of the nucleotide sequences of the H protein and Fprotein:

The nucleotide sequences of the H and F proteins are determined inaccordance with the method of Isegawa et al. (Mol. Cell. Prob., 6,467-475, 1992). Each of the measles strains shown in Tables 2 and 3 isinfected individually to B95a cells, and RNA is extracted from each ofthe infected cells by GTC/CsCl method of Chirgwin et al. (Biochemistry,18, 5294-5299, 1979). Subsequently, the cDNA for each measles strain issynthesized using random primers (6 mer). Based on the cDNA sequence ofthe gene of Edmonston strain (Virology, vol. 173, no. 2, pp. 415-425,1989), specific primers (shown in Table 1) are synthesized, and thenucleotide sequence of each of the measles strains is determined by thePCR-direct sequencing method using the synthesized primers. The aminoacid sequence is deduced from each nucleotide sequence in accordancewith the universal code, and in addition, the amino acids which aresubstituted as a result of genetic mutations are identified. The resultsare shown in Tables 2 and 3 and SEQ ID NOs: 1, 2, 9, 10, and 17 to 20.

TABLE 1 Primers used for PCR-direct sequencing method Primer Nucleicacid sequence Gene coding for F28(SEQ ID NO:21) AGAATCAAGACTCATCCAATGTCF protein CF7(SEQ ID NO:22) TTGAGAGTTCAGCATGGACTGGT CF3(SEQ ID NO:23)ACAATGAAGTAGGACTCTGTGTC F3(SEQ ID NO:24) GGAACCTAATAGCCAATTGTGCA CF2(SEQID NO:25) CGAGGTCAATTCTGTGCAAGTAC F4(SEQ ID NO:26)AAAGGGAGAACAAGTTGGTATGT CF1(SEQ ID NO:27) GATATTGTTCGGCCAGAGGGAAG Genecoding for MP5(SEQ ID NO:28) ATGTCACCACAACGAGACCGGAT H protein MP4(SEQID NO:29) GAGATTCACTGACCTAGTGAAAT MP2(SEQ ID NO:30)TCGCTGTCCCTGTTAGACTTGTA H8(SEQ ID NO:31) GAGCAACCAGTCAGTAATGATCT MP3(SEQID NO:32) ATGCCTGATGTCTGGGTGACATC

TABLE 2 Amino acid substitutions in H protein Amino acid Measles strainnumber Edmo Tana CAM-70 F-b F-t U-b U-t MO NA  93 Thr Ile 157 Val Ala169 Ser Ala Ala Ala Ala Ala Ala 174 Thr Ala Ala Ala Ala Ala Ala 175 ArgLys Lys 176 Thr Ala Ala Ala Ala Ala Ala 211 Gly Ser Ser Ser Ser Ser Ser243 Arg Gly Gly Gly Gly Gly Gly 252 Tyr His His His His His His 276 LeuPhe Phe Phe Phe Phe Phe 279 Pro Ser Ser Ser Ser Ser Ser 284 Leu Phe PhePhe Phe Phe Phe 285 Ser Asn Asn 296 Leu Phe Phe Phe Phe Phe Phe 302 GlyArg Arg Arg Arg Arg Arg 316 Gly Ser Ser Ser Ser 338 Pro Ser 387 Leu Gln416 Asp Asn Asn Asn Asn Asn Asn 455 Thr Asn 481 Tyr Asn Asn Asn Asn AsnAsn 484 Asn Thr Thr Thr Thr Thr Thr Thr Thr 505 Asp Gly 546 Ser Gly 592Gly Glu Glu 600 Glu Val Val Val Val Val Val Val Val 603 Gly Glu 616 ArgSer Ser Ser Ser Ser Ser [Note] (1) “Edmo” represents “Edmonston strain”,“Tana” represents “Tanabe strain”, and “MO” represents “Momo strain”.(2) Amino acid sequence of H protein (deduced from cDNA) of Edmonstonstrain is used as a standard for determining the substituted amino acidsin H protein of other measles strains. Amino acids which are the same asthat of the Edmonston strain are not shown.

TABLE 3 Amino acid substitutions in F protein Amino acid Measles strainnumber Edmo Tana CAM-70 F-b F-t U-b U-t MO NA  11 Phe Ile Leu Leu  52Gln His His 107 Ser Gly Gly 165 Arg Gly 398 tyr His 417 Ala Asp Asp 523Lys Arg Arg Arg Arg Arg Arg [Note] (1) “Edmo” represents “Edmonstonstrain”, “Tana” represents “Tanabe strain”, and “MO” represents “Momostrain”. (2) Amino acid sequence of F protein (deduced from cDNA) ofEdmonston strain is used as a standard for determining the substitutedamino acids in F protein of other measles strains. Amino acids which arethe same as that of the Edmonston strain are not shown.

Determination of the Secondary Structure of the H Protein:

The secondary structure of the H protein is determined by analyzing theabove-identified amino acid sequence by computer. Computer software“DNASIS-Mac (version 3.6)” (manufactured and sold by Hitachi SoftwareEngineering Co., Ltd., Japan) is used to analyze the hydrophobicitypattern and to conduct Chou-Fasman analysis. As a result, with respectto the secondary structure of each of the regions respectivelyconsisting of the 176th to 316th amino acids and the 317th to 616thamino acids of the whole amino acid sequence of the H protein shown inSEQ ID NO: 2, the positions of epitopes are flip-flopped between thevaccine strain and the epidemic strain [that is, when an analyticaldiagram for a vaccine strain (for example, CAM-70 strain) and ananalytical diagram for an epidemic strain (for example, MO strain or NAstrain) are arranged side by side, it is apparent that the diagram forthe epidemic strain is transformed to look like a mirror image (axialsymmetry) of the diagram for the vaccine strain]. On the other hand,with respect to the F protein, when the analytical diagrams are preparedfor a vaccine strain and an epidemic strain, no such differences aswould cause a mirror image (axial symmetry) are observed.

Analysis of the Mutated Epitopes of the H Protein:

With respect to the amino acid sequences of the above-mentioned vaccinestrain and epidemic strain, the regions where the mutation (amino acidsubstitutions) is concentrated are analyzed by computer using thecomputer software “Epitope Advisor” [manufactured and sold by FujitsuKyushu System Engineering (FQS) Co., Ltd., Japan] to determine theepitopes. As a result, the following four regions, the 172nd to 178thamino acids, the 238th to 244th amino acids, the 277th to 282nd aminoacids, and the 301st to 307th amino acids, identified with thepositional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10,are determined as the mutated epitope regions of the H protein.

EXAMPLE 2

Modification of a Genome of a Live Attenuated Vaccine Strain:

A recombinant CAM-70 virus, which is vaccine virus CAM-70 strain havinga part of its H protein replaced by the corresponding part of the Hprotein of epidemic measles Momo strain, is prepared by the method ofRadecke et al. (reverse genetics) described in item [II](1) above.

The part of the H protein to be replaced is the restriction enzyme HinfIfragment of the cDNA derived from the viral genome comprising the regionconsisting of the 526th to 948th nucleotides (total of 423 nucleotides)of the nucleotide sequence of SEQ ID NO: 1 (encoding the 176th to 316thamino acids of H protein). The antigenicity of the prepared recombinantvirus is confirmed by the fluorescent antibody technique and the enzymeimmunoassay using the monoclonal antibodies against CAM-70 strain andMomo strain.

EXAMPLE 3

Modification of a Genome of a Live Attenuated Vaccine Strain:

A recombinant CAM-70 virus which is vaccine virus CAM-70 strain having apart of its H protein replaced by the corresponding part of the Hprotein of epidemic measles NA strain is prepared in substantially thesame manner as mentioned in Example 2, except that the method ismodified in the following manner {modified method described in item[II](1) above}.

First, the viral genomic RNA is extracted from the CAM-70 strain, andthe cDNA is prepared from the genomic RNA by RT-PCR (reversetranscript-PCR) method. The genes coding for N, P and L proteins arecloned individually from the prepared cDNA by a customary method usingprimers [hereinbelow, each of the clones are referred to as“pcDNA3.1(−)/N”, “pcDNA3.1(−)/P” and “pcDNA3.1(−)/L”]. The clones areamplified in E. Coli and stored for use in the subsequent procedure.

In addition to the above, the cDNA derived from the full length viralgenomic RNA of CAM-70 strain, in which a part of its H protein gene isreplaced by the corresponding part of the H protein gene of NA strain,is cloned by using plasmids pbluescript SK or KS in substantially thesame manner as mentioned above, to thereby obtain clone pBluescript/MV.The obtained clone is amplified and stored for use in the subsequentprocedure. With respect to the nucleotide sequence replaced in the cDNAfor CAM-70 strain, the region consisting of the 526th to 948thnucleotides (total of 423 nucleotides encoding the 176th to 316th aminoacids) of the CAM-70 strain H protein gene of SEQ ID NO: 1 is replacedwith the corresponding region (the 526th to 948th nucleotides) in the NAstrain H protein gene of SEQ ID NO: 9.

The above-mentioned pcDNA3.1(−)/N, pcDNA3.1(−)/P, pcDNA3.1(−)/L andpBluescript/MV are co-transfected to MRC-5 cells which have already beentransfected with recMVA (FEBS Letter, vol. 371, no. 1, pp. 9-12, 1995),and then, the transfected cells are cultured at 37° C. to thereby obtaina recombinant virus. The antigenicity of the recombinant virus isconfirmed by the fluorescent antibody technique and the enzymeimmunoassay using the monoclonal antibodies against CAM-70 strain and NAstrain. The antigenicity of the recombinant virus is on the same levelas that of the epidemic strains, and since the recombinant virus isattenuated, it can be used as an active component for a live attenuatedmeasles vaccine.

EXAMPLE 4

Preparation of an Active Component for a Gene Vaccine:

A non-proliferating recombinant adenovirus is prepared in accordancewith the method of Saito et al. described in item [II](2) above. Withrespect to the gene which is inserted into the non-proliferating viralgenome (i.e., cassette cosmid pAdex1), use is made of a ligation productof the cDNAs for the H protein gene and F protein gene of NA strainrespectively shown in SEQ ID NO: 9 and SEQ ID NO: 19. The two cDNAs areligated in the order of F protein-H protein in the direction from the 5′end to the 3′ end, so that the F protein and the H protein are expressedin the form of an F-H fusion protein. The ligated cDNA is inserted intothe E1A·E1B deletion site of the cassette cosmid pAdex1 cleaved with arestriction enzyme SwaI, to thereby obtain pAdex1/FH.

With respect to the cDNAs for the H protein gene and the F protein gene,the cDNAs are prepared from the NA strain genomic RNA by RT-PCR methodusing primers which correspond to the respective genes. Further,pAdex1/FH is packaged into a λphage so as to be amplified in E. coli,and stored for use in the subsequent procedure.

Subsequently, the DNA-TPC (viral DNA-Terminal Protein Complex) of theparent adenovirus strain is extracted and purified from the infectedcells by CsCl ultracentrifugation method, and the purified DNA-TPC isdigested with restriction enzyme Eco T22I to thereby obtain thedigestion product DNA-TPC/Eco T22I. Then, using calcium phosphatemethod, the above-obtained pAdex1/FH and DNA-TPC/Eco T221 areco-transfected to the cultured cells of 293 cell line, thereby obtainingtransfectants, followed by culturing the transfectants at 37° C. for 18hours to advance the homologous recombination between the DNAs. As aresult of the homologous recombination, a non-proliferating recombinantadenovirus having both the H and F proteins of NA strain is obtainedfrom the cultured transfectants.

The fluorescent antibody technique using monoclonal antibodies againsteach of the H and F proteins is conducted to select the recombinantvirus and confirm its proliferation in the transfected cells of 293 cellline (which are the permissive host cells for the adenovirus).

EXAMPLE 5

Preparation of a Diagnostic Reagent:

The peptides having the following amino acid sequences are synthesizedusing a peptide synthesizer (Model ABI 432A manufactured and sold byPerkin-Elmer Cetus Co., Ltd., U.S.A.): “Leu Glu Ala Arg Ala Thr Asn”,“Asn Leu Ser Ser Lys Gly Ser”, “Glu Gln Ser Val Ser Asn” and “His ArgGlu Asp Ser Ile Thr”. Each of the synthesized peptides is used as anantigen for recognizing and identifying the infection with the epidemicstrains.

INDUSTRIAL APPLICABILITY

By the use of the measles virus mutant antigen or the gene coding forthe same of the present invention, it has become possible to provideefficiently and economically a live attenuated measles vaccine or genevaccine which is adapted for an epidemic strain of measles virus, and adiagnostic reagent capable of accurately detecting infections with anepidemic strain of measles virus.

32 1 1854 DNA Measles virus CDS (1)..(1851) Attenuated measles virusCAM-70 strain 1 atg tca cca caa cga gac cgg ata aat gcc ttc tac aaa gataac ccc 48 Met Ser Pro Gln Arg Asp Arg Ile Asn Ala Phe Tyr Lys Asp AsnPro 1 5 10 15 cat ccc aag gga agt agg ata gtc att aac aga gaa cat cttatg att 96 His Pro Lys Gly Ser Arg Ile Val Ile Asn Arg Glu His Leu MetIle 20 25 30 gat aga cct tat gtt ttg ctg gct gtt ctg ttt gtc atg ttt ctgagc 144 Asp Arg Pro Tyr Val Leu Leu Ala Val Leu Phe Val Met Phe Leu Ser35 40 45 ttg atc ggg ttg cta gcc att gca ggc att aga ctt cat cgg gca gcc192 Leu Ile Gly Leu Leu Ala Ile Ala Gly Ile Arg Leu His Arg Ala Ala 5055 60 atc tac acc gca gag atc cat aaa agc ctc agc acc aat cta gat gta240 Ile Tyr Thr Ala Glu Ile His Lys Ser Leu Ser Thr Asn Leu Asp Val 6570 75 80 act aac tca atc gag cat cag gtc aag gac gtg ctg ata cca ctc ttc288 Thr Asn Ser Ile Glu His Gln Val Lys Asp Val Leu Ile Pro Leu Phe 8590 95 aaa atc atc ggt gat gaa gtg ggc ctg agg aca cct cag aga ttc act336 Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro Gln Arg Phe Thr 100105 110 gac cta gtg aaa ttc atc tct gac aag att aaa ttc ctt aat ccg gat384 Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys Phe Leu Asn Pro Asp 115120 125 agg gag tac gac ttc aga gat ctc act tgg tgt atc aac ccg cca gag432 Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp Cys Ile Asn Pro Pro Glu 130135 140 aga atc aaa ttg gat tat gat caa tac tgt gca gat gcg gct gct gaa480 Arg Ile Lys Leu Asp Tyr Asp Gln Tyr Cys Ala Asp Ala Ala Ala Glu 145150 155 160 gag ctc atg aat gca ttg gtg aac tca act cta ctg gag acc aaaaca 528 Glu Leu Met Asn Ala Leu Val Asn Ser Thr Leu Leu Glu Thr Lys Thr165 170 175 acc aat cag ttc cta gct gtc tca aag gga aac tgc tca ggg cccact 576 Thr Asn Gln Phe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr180 185 190 aca atc aga ggt caa ttc tca aac atg tcg ctg tcc ctg tta gacttg 624 Thr Ile Arg Gly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu195 200 205 tat tta ggt cga ggt tac aat gtg tca tct ata gtc act atg acatcc 672 Tyr Leu Gly Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser210 215 220 cag gga atg tat ggg gga act tac cta gtg gaa aag cct aat ctgagc 720 Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser225 230 235 240 agc aaa agg tca gag ttg tca caa ctg agc atg tac cga gtgttt gaa 768 Ser Lys Arg Ser Glu Leu Ser Gln Leu Ser Met Tyr Arg Val PheGlu 245 250 255 gta ggt gtt atc aga aat ccg ggt ttg ggg gct ccg gtg ttccat atg 816 Val Gly Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe HisMet 260 265 270 aca aac tat ctt gag caa cca gtc agt aat gat ctc agc aactgt atg 864 Thr Asn Tyr Leu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn CysMet 275 280 285 gtg gct ttg ggg gag ctc aaa ctc gca gcc ctt tgt cac ggggaa gat 912 Val Ala Leu Gly Glu Leu Lys Leu Ala Ala Leu Cys His Gly GluAsp 290 295 300 tct atc aca att ccc tat cag gga tca ggg aaa ggt gtc agcttc cag 960 Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser PheGln 305 310 315 320 ctc gtc aag cta ggt gtc tgg aaa tcc cca acc gac atgcaa tcc tgg 1008 Leu Val Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met GlnSer Trp 325 330 335 gtc tcc tta tca acg gat gat cca gtg ata gac agg ctttac ctc tca 1056 Val Ser Leu Ser Thr Asp Asp Pro Val Ile Asp Arg Leu TyrLeu Ser 340 345 350 tct cac aga ggt gtt atc gct gac aat caa gca aaa tgggct gtc ccg 1104 Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala Lys Trp AlaVal Pro 355 360 365 aca aca cga aca gat gac aag ttg cga atg gag aca tgcttc caa cag 1152 Thr Thr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr Cys PheGln Gln 370 375 380 gcg tgt aag ggt aaa atc caa gca ctc tgc gag aat cccgag tgg gca 1200 Ala Cys Lys Gly Lys Ile Gln Ala Leu Cys Glu Asn Pro GluTrp Ala 385 390 395 400 cca ttg aag gat aac agg att cct tca tac ggg gtcttg tct gtt gat 1248 Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val LeuSer Val Asp 405 410 415 ctg agt ctg aca gtt gag ctt aaa atc aaa att gcttcg gga ttc ggg 1296 Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile Ala SerGly Phe Gly 420 425 430 cca ttg atc aca cac ggt tca ggg atg gac cta tacaaa tcc aac cac 1344 Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu Tyr LysSer Asn His 435 440 445 aac aat gtg tat tgg ctg aat atc ccg cca atg aagaac cta gcc tta 1392 Asn Asn Val Tyr Trp Leu Asn Ile Pro Pro Met Lys AsnLeu Ala Leu 450 455 460 ggt gta atc aac aca ttg gag tgg ata ccg aga ttcaag gtt agc ccc 1440 Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe LysVal Ser Pro 465 470 475 480 tac ctc ttc act gtc cca att aag gaa gca ggcgaa gac tgc cat gcc 1488 Tyr Leu Phe Thr Val Pro Ile Lys Glu Ala Gly GluAsp Cys His Ala 485 490 495 cca aca tac cta cct gcg gag gtg ggt ggt gatgtc aaa ctc agt tcc 1536 Pro Thr Tyr Leu Pro Ala Glu Val Gly Gly Asp ValLys Leu Ser Ser 500 505 510 aat ctg gtg att cta cct ggt caa gat ctc caatat gtt ttg gca acc 1584 Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln TyrVal Leu Ala Thr 515 520 525 tac gat act tcc agg gtt gaa cat gct gtg gtttat tac gtt tac agc 1632 Tyr Asp Thr Ser Arg Val Glu His Ala Val Val TyrTyr Val Tyr Ser 530 535 540 cca agc cgc tca ttt tct tac ttt tat cct tttagg ttg cct ata aag 1680 Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe ArgLeu Pro Ile Lys 545 550 555 560 ggg gtc ccc atc gaa tta caa gtg gaa tgcttc aca tgg gac caa aaa 1728 Gly Val Pro Ile Glu Leu Gln Val Glu Cys PheThr Trp Asp Gln Lys 565 570 575 ctc tgg tgc cgt cac ttc tgt gtg ctt gcggac tca gaa tct ggt gaa 1776 Leu Trp Cys Arg His Phe Cys Val Leu Ala AspSer Glu Ser Gly Glu 580 585 590 cat atc act cac tct ggg atg gtg ggc atggaa gtc agc tgc aca gtc 1824 His Ile Thr His Ser Gly Met Val Gly Met GluVal Ser Cys Thr Val 595 600 605 acc cgg gaa gat gga acc aat cgc aga tag1854 Thr Arg Glu Asp Gly Thr Asn Arg Arg 610 615 2 617 PRT Measles virusany n or Xaa = Unknown 2 Met Ser Pro Gln Arg Asp Arg Ile Asn Ala Phe TyrLys Asp Asn Pro 1 5 10 15 His Pro Lys Gly Ser Arg Ile Val Ile Asn ArgGlu His Leu Met Ile 20 25 30 Asp Arg Pro Tyr Val Leu Leu Ala Val Leu PheVal Met Phe Leu Ser 35 40 45 Leu Ile Gly Leu Leu Ala Ile Ala Gly Ile ArgLeu His Arg Ala Ala 50 55 60 Ile Tyr Thr Ala Glu Ile His Lys Ser Leu SerThr Asn Leu Asp Val 65 70 75 80 Thr Asn Ser Ile Glu His Gln Val Lys AspVal Leu Ile Pro Leu Phe 85 90 95 Lys Ile Ile Gly Asp Glu Val Gly Leu ArgThr Pro Gln Arg Phe Thr 100 105 110 Asp Leu Val Lys Phe Ile Ser Asp LysIle Lys Phe Leu Asn Pro Asp 115 120 125 Arg Glu Tyr Asp Phe Arg Asp LeuThr Trp Cys Ile Asn Pro Pro Glu 130 135 140 Arg Ile Lys Leu Asp Tyr AspGln Tyr Cys Ala Asp Ala Ala Ala Glu 145 150 155 160 Glu Leu Met Asn AlaLeu Val Asn Ser Thr Leu Leu Glu Thr Lys Thr 165 170 175 Thr Asn Gln PheLeu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr 180 185 190 Thr Ile ArgGly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu 195 200 205 Tyr LeuGly Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser 210 215 220 GlnGly Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser 225 230 235240 Ser Lys Arg Ser Glu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe Glu 245250 255 Val Gly Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met260 265 270 Thr Asn Tyr Leu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn CysMet 275 280 285 Val Ala Leu Gly Glu Leu Lys Leu Ala Ala Leu Cys His GlyGlu Asp 290 295 300 Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly ValSer Phe Gln 305 310 315 320 Leu Val Lys Leu Gly Val Trp Lys Ser Pro ThrAsp Met Gln Ser Trp 325 330 335 Val Ser Leu Ser Thr Asp Asp Pro Val IleAsp Arg Leu Tyr Leu Ser 340 345 350 Ser His Arg Gly Val Ile Ala Asp AsnGln Ala Lys Trp Ala Val Pro 355 360 365 Thr Thr Arg Thr Asp Asp Lys LeuArg Met Glu Thr Cys Phe Gln Gln 370 375 380 Ala Cys Lys Gly Lys Ile GlnAla Leu Cys Glu Asn Pro Glu Trp Ala 385 390 395 400 Pro Leu Lys Asp AsnArg Ile Pro Ser Tyr Gly Val Leu Ser Val Asp 405 410 415 Leu Ser Leu ThrVal Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly 420 425 430 Pro Leu IleThr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His 435 440 445 Asn AsnVal Tyr Trp Leu Asn Ile Pro Pro Met Lys Asn Leu Ala Leu 450 455 460 GlyVal Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro 465 470 475480 Tyr Leu Phe Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala 485490 495 Pro Thr Tyr Leu Pro Ala Glu Val Gly Gly Asp Val Lys Leu Ser Ser500 505 510 Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu AlaThr 515 520 525 Tyr Asp Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr ValTyr Ser 530 535 540 Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg LeuPro Ile Lys 545 550 555 560 Gly Val Pro Ile Glu Leu Gln Val Glu Cys PheThr Trp Asp Gln Lys 565 570 575 Leu Trp Cys Arg His Phe Cys Val Leu AlaAsp Ser Glu Ser Gly Glu 580 585 590 His Ile Thr His Ser Gly Met Val GlyMet Glu Val Ser Cys Thr Val 595 600 605 Thr Arg Glu Asp Gly Thr Asn ArgArg 610 615 3 524 PRT Measles virus Attenuated measles virus CAM-70strain 3 Ile Pro Leu Phe Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro1 5 10 15 Gln Arg Phe Thr Asp Leu Val Lys Phe Ile Ser Asp Lys Ile LysPhe 20 25 30 Leu Asn Pro Asp Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp CysIle 35 40 45 Asn Pro Pro Glu Arg Ile Lys Leu Asp Tyr Asp Gln Tyr Cys AlaAsp 50 55 60 Ala Ala Ala Glu Glu Leu Met Asn Ala Leu Val Asn Ser Thr LeuLeu 65 70 75 80 Glu Thr Lys Thr Thr Asn Gln Phe Leu Ala Val Ser Lys GlyAsn Cys 85 90 95 Ser Gly Pro Thr Thr Ile Arg Gly Gln Phe Ser Asn Met SerLeu Ser 100 105 110 Leu Leu Asp Leu Tyr Leu Gly Arg Gly Tyr Asn Val SerSer Ile Val 115 120 125 Thr Met Thr Ser Gln Gly Met Tyr Gly Gly Thr TyrLeu Val Glu Lys 130 135 140 Pro Asn Leu Ser Ser Lys Arg Ser Glu Leu SerGln Leu Ser Met Tyr 145 150 155 160 Arg Val Phe Glu Val Gly Val Ile ArgAsn Pro Gly Leu Gly Ala Pro 165 170 175 Val Phe His Met Thr Asn Tyr LeuGlu Gln Pro Val Ser Asn Asp Leu 180 185 190 Ser Asn Cys Met Val Ala LeuGly Glu Leu Lys Leu Ala Ala Leu Cys 195 200 205 His Gly Glu Asp Ser IleThr Ile Pro Tyr Gln Gly Ser Gly Lys Gly 210 215 220 Val Ser Phe Gln LeuVal Lys Leu Gly Val Trp Lys Ser Pro Thr Asp 225 230 235 240 Met Gln SerTrp Val Ser Leu Ser Thr Asp Asp Pro Val Ile Asp Arg 245 250 255 Leu TyrLeu Ser Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala Lys 260 265 270 TrpAla Val Pro Thr Thr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr 275 280 285Cys Phe Gln Gln Ala Cys Lys Gly Lys Ile Gln Ala Leu Cys Glu Asn 290 295300 Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val 305310 315 320 Leu Ser Val Asp Leu Ser Leu Thr Val Glu Leu Lys Ile Lys IleAla 325 330 335 Ser Gly Phe Gly Pro Leu Ile Thr His Gly Ser Gly Met AspLeu Tyr 340 345 350 Lys Ser Asn His Asn Asn Val Tyr Trp Leu Asn Ile ProPro Met Lys 355 360 365 Asn Leu Ala Leu Gly Val Ile Asn Thr Leu Glu TrpIle Pro Arg Phe 370 375 380 Lys Val Ser Pro Tyr Leu Phe Thr Val Pro IleLys Glu Ala Gly Glu 385 390 395 400 Asp Cys His Ala Pro Thr Tyr Leu ProAla Glu Val Gly Gly Asp Val 405 410 415 Lys Leu Ser Ser Asn Leu Val IleLeu Pro Gly Gln Asp Leu Gln Tyr 420 425 430 Val Leu Ala Thr Tyr Asp ThrSer Arg Val Glu His Ala Val Val Tyr 435 440 445 Tyr Val Tyr Ser Pro SerArg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg 450 455 460 Leu Pro Ile Lys GlyVal Pro Ile Glu Leu Gln Val Glu Cys Phe Thr 465 470 475 480 Trp Asp GlnLys Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp Ser 485 490 495 Glu SerGly Glu His Ile Thr His Ser Gly Met Val Gly Met Glu Val 500 505 510 SerCys Thr Val Thr Arg Glu Asp Gly Thr Asn Arg 515 520 4 141 PRT Measlesvirus Attenuated measles virus CAM-70 strain 4 Thr Thr Asn Gln Phe LeuAla Val Ser Lys Gly Asn Cys Ser Gly Pro 1 5 10 15 Thr Thr Ile Arg GlyGln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp 20 25 30 Leu Tyr Leu Gly ArgGly Tyr Asn Val Ser Ser Ile Val Thr Met Thr 35 40 45 Ser Gln Gly Met TyrGly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu 50 55 60 Ser Ser Lys Arg SerGlu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe 65 70 75 80 Glu Val Gly ValIle Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His 85 90 95 Met Thr Asn TyrLeu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn Cys 100 105 110 Met Val AlaLeu Gly Glu Leu Lys Leu Ala Ala Leu Cys His Gly Glu 115 120 125 Asp SerIle Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly 130 135 140 5 7 PRT Measlesvirus Attenuated measles virus CAM-70 strain 5 Leu Glu Thr Lys Thr ThrAsn 1 5 6 7 PRT Measles virus Attenuated measles virus CAM-70 strain 6Asn Leu Ser Ser Lys Arg Ser 1 5 7 6 PRT Measles virus Attenuated measlesvirus CAM -70 strain 7 Glu Gln Pro Val Ser Asn 1 5 8 7 PRT Measles virusAttenuated measles virus CAM -70 strain 8 His Gly Glu Asp Ser Ile Thr 15 9 1854 DNA Measles virus CDS (1)..(1851) Attenuated measles virus NAstrain 9 atg tca cca caa cga gac cga ata aat gcc ttc tac aaa gac aac ccc48 Met Ser Pro Gln Arg Asp Arg Ile Asn Ala Phe Tyr Lys Asp Asn Pro 1 510 15 cat cct aag gga agt agg ata gtt att aac aga gaa cat ctt atg att 96His Pro Lys Gly Ser Arg Ile Val Ile Asn Arg Glu His Leu Met Ile 20 25 30gat aga cct tat gtt ttg ctg gct gtt cta ttc gtc atg ttt ctg agc 144 AspArg Pro Tyr Val Leu Leu Ala Val Leu Phe Val Met Phe Leu Ser 35 40 45 ttgatc ggg ttg cta gcc att gca ggc att aga ctt cat cgg gca gcc 192 Leu IleGly Leu Leu Ala Ile Ala Gly Ile Arg Leu His Arg Ala Ala 50 55 60 atc tacact gca gag atc cat aaa agc ctc agc acc aat cta gat gta 240 Ile Tyr ThrAla Glu Ile His Lys Ser Leu Ser Thr Asn Leu Asp Val 65 70 75 80 act aactca atc gag cat cag gtc aag gac gtg ctg aca cca ctc ttc 288 Thr Asn SerIle Glu His Gln Val Lys Asp Val Leu Thr Pro Leu Phe 85 90 95 aag atc atcggt gat gaa gtg ggc ctg agg aca cct cag aga ttc act 336 Lys Ile Ile GlyAsp Glu Val Gly Leu Arg Thr Pro Gln Arg Phe Thr 100 105 110 gac cta gtgaaa ttc atc tct gac aag att aaa ttc ctt aat ccg gat 384 Asp Leu Val LysPhe Ile Ser Asp Lys Ile Lys Phe Leu Asn Pro Asp 115 120 125 agg gag tacgac ttc agg gat ctc act tgg tgt atc aac ccg cca gag 432 Arg Glu Tyr AspPhe Arg Asp Leu Thr Trp Cys Ile Asn Pro Pro Glu 130 135 140 aga atc aaattg gat tat gat caa tac tgt gca gat gtg gct gct gaa 480 Arg Ile Lys LeuAsp Tyr Asp Gln Tyr Cys Ala Asp Val Ala Ala Glu 145 150 155 160 gaa ctcatg aat gca ttg gtg aac gca act cta ctg gag gcc agg gca 528 Glu Leu MetAsn Ala Leu Val Asn Ala Thr Leu Leu Glu Ala Arg Ala 165 170 175 acc aatcag ttc cta gct gtc tca aag gga aac tgc tca ggg ccc act 576 Thr Asn GlnPhe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr 180 185 190 aca atcaga ggt caa ttc tca aac atg tcg ctg tcc ctg ttg gac ttg 624 Thr Ile ArgGly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu 195 200 205 tac ttaagt cga ggt tac aat gtg tca tct ata gtc act atg aca tcc 672 Tyr Leu SerArg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser 210 215 220 cag ggaatg tac ggg gga act tac cta gtg gaa aag cct aat ctg agc 720 Gln Gly MetTyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser 225 230 235 240 agtaaa ggg tca gag ttg tca caa ctg agc atg cac cga gtg ttt gaa 768 Ser LysGly Ser Glu Leu Ser Gln Leu Ser Met His Arg Val Phe Glu 245 250 255 gtaggt gtg atc aga aat ccg ggt ttg ggg gct ccg gtg ttc cat atg 816 Val GlyVal Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met 260 265 270 acgaac tat ttt gag caa tca gtc agt aat gat ttc aac aac tgc atg 864 Thr AsnTyr Phe Glu Gln Ser Val Ser Asn Asp Phe Asn Asn Cys Met 275 280 285 gtggct ttg ggg gag ctc aaa ttc gca gcc ctc tgt cac agg gaa gat 912 Val AlaLeu Gly Glu Leu Lys Phe Ala Ala Leu Cys His Arg Glu Asp 290 295 300 tctatc aca att ccc tat cag ggg tca ggg aaa ggt gtc agc ttc cag 960 Ser IleThr Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln 305 310 315 320ctc gtc aag cta ggt gtc tgg aaa tcc cca acc gac atg caa tcc tgg 1008 LeuVal Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met Gln Ser Trp 325 330 335gtc ccc cta tca acg gat gat cca gtg ata gat agg ctt tac ctc tca 1056 ValPro Leu Ser Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser 340 345 350tct cac aga ggt gtt atc gct gac aat caa gca aaa tgg gct gtc ccg 1104 SerHis Arg Gly Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro 355 360 365aca aca cga aca gat gac aag ttg cga atg gag aca tgc ttc cag cag 1152 ThrThr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln 370 375 380gcg tgt cag ggc aaa atc caa gca ctc tgc gag aat ccc gag tgg gca 1200 AlaCys Gln Gly Lys Ile Gln Ala Leu Cys Glu Asn Pro Glu Trp Ala 385 390 395400 cca ctg aag gac aac agg att cct tca tac ggg gtc ttg tct gtt aat 1248Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asn 405 410415 ctg agt ctg aca gtt gag ctc aaa atc aaa att gct tca gga ttc ggg 1296Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly 420 425430 cca ttg atc aca cac ggt tca ggg atg gac cta tac aaa tcc aac cac 1344Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His 435 440445 aac aat gtg tat tgg ctg acc atc ccg cca atg aag aac cta gcc tta 1392Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu 450 455460 ggt gta atc aac aca tta gag tgg ata ccg aga ttc aag gtt agt ccc 1440Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro 465 470475 480 aac ctc ttc act gtt cca atc aag gaa gca ggc gag gac tgc cat gcc1488 Asn Leu Phe Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala 485490 495 cca aca tac ctg cct gcg gag gtg gat ggt gat gtc aaa ctc agt tcc1536 Pro Thr Tyr Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser 500505 510 aat ctg gtg att cta cct ggt caa gat ctc caa tat gtt ttg gca acc1584 Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr 515520 525 tac gat act tcc agg gtt gaa cat gct gtg gtt tat tat gtt tac agc1632 Tyr Asp Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser 530535 540 ccg agc cgc tca ttt tct tac ttt tat ccc ttt agg ttg cct ata aag1680 Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys 545550 555 560 ggg gtc ccc atc gaa tta caa gtg gaa tgc ttc aca tgg gac caaaaa 1728 Gly Val Pro Ile Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys565 570 575 ctc tgg tgc cgt cac ttc tgt gtg ctt gcg gac tca gaa tct ggtgga 1776 Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly580 585 590 cat atc act cac tct gga atg gtg ggc atg gga gtc agc tgc acagtc 1824 His Ile Thr His Ser Gly Met Val Gly Met Gly Val Ser Cys Thr Val595 600 605 act cgg gaa gat gga acc aat agc aga tag 1854 Thr Arg Glu AspGly Thr Asn Ser Arg 610 615 10 617 PRT Measles virus any n or Xaa =Unknown 10 Met Ser Pro Gln Arg Asp Arg Ile Asn Ala Phe Tyr Lys Asp AsnPro 1 5 10 15 His Pro Lys Gly Ser Arg Ile Val Ile Asn Arg Glu His LeuMet Ile 20 25 30 Asp Arg Pro Tyr Val Leu Leu Ala Val Leu Phe Val Met PheLeu Ser 35 40 45 Leu Ile Gly Leu Leu Ala Ile Ala Gly Ile Arg Leu His ArgAla Ala 50 55 60 Ile Tyr Thr Ala Glu Ile His Lys Ser Leu Ser Thr Asn LeuAsp Val 65 70 75 80 Thr Asn Ser Ile Glu His Gln Val Lys Asp Val Leu ThrPro Leu Phe 85 90 95 Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro GlnArg Phe Thr 100 105 110 Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys PheLeu Asn Pro Asp 115 120 125 Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp CysIle Asn Pro Pro Glu 130 135 140 Arg Ile Lys Leu Asp Tyr Asp Gln Tyr CysAla Asp Val Ala Ala Glu 145 150 155 160 Glu Leu Met Asn Ala Leu Val AsnAla Thr Leu Leu Glu Ala Arg Ala 165 170 175 Thr Asn Gln Phe Leu Ala ValSer Lys Gly Asn Cys Ser Gly Pro Thr 180 185 190 Thr Ile Arg Gly Gln PheSer Asn Met Ser Leu Ser Leu Leu Asp Leu 195 200 205 Tyr Leu Ser Arg GlyTyr Asn Val Ser Ser Ile Val Thr Met Thr Ser 210 215 220 Gln Gly Met TyrGly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser 225 230 235 240 Ser LysGly Ser Glu Leu Ser Gln Leu Ser Met His Arg Val Phe Glu 245 250 255 ValGly Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met 260 265 270Thr Asn Tyr Phe Glu Gln Ser Val Ser Asn Asp Phe Asn Asn Cys Met 275 280285 Val Ala Leu Gly Glu Leu Lys Phe Ala Ala Leu Cys His Arg Glu Asp 290295 300 Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln305 310 315 320 Leu Val Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met GlnSer Trp 325 330 335 Val Pro Leu Ser Thr Asp Asp Pro Val Ile Asp Arg LeuTyr Leu Ser 340 345 350 Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala LysTrp Ala Val Pro 355 360 365 Thr Thr Arg Thr Asp Asp Lys Leu Arg Met GluThr Cys Phe Gln Gln 370 375 380 Ala Cys Gln Gly Lys Ile Gln Ala Leu CysGlu Asn Pro Glu Trp Ala 385 390 395 400 Pro Leu Lys Asp Asn Arg Ile ProSer Tyr Gly Val Leu Ser Val Asn 405 410 415 Leu Ser Leu Thr Val Glu LeuLys Ile Lys Ile Ala Ser Gly Phe Gly 420 425 430 Pro Leu Ile Thr His GlySer Gly Met Asp Leu Tyr Lys Ser Asn His 435 440 445 Asn Asn Val Tyr TrpLeu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu 450 455 460 Gly Val Ile AsnThr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro 465 470 475 480 Asn LeuPhe Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala 485 490 495 ProThr Tyr Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser 500 505 510Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr 515 520525 Tyr Asp Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser 530535 540 Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys545 550 555 560 Gly Val Pro Ile Glu Leu Gln Val Glu Cys Phe Thr Trp AspGln Lys 565 570 575 Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp Ser GluSer Gly Gly 580 585 590 His Ile Thr His Ser Gly Met Val Gly Met Gly ValSer Cys Thr Val 595 600 605 Thr Arg Glu Asp Gly Thr Asn Ser Arg 610 61511 524 PRT Measles virus Attenuated measles virus NA strain 11 Thr ProLeu Phe Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro 1 5 10 15 GlnArg Phe Thr Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys Phe 20 25 30 LeuAsn Pro Asp Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp Cys Ile 35 40 45 AsnPro Pro Glu Arg Ile Lys Leu Asp Tyr Asp Gln Tyr Cys Ala Asp 50 55 60 ValAla Ala Glu Glu Leu Met Asn Ala Leu Val Asn Ala Thr Leu Leu 65 70 75 80Glu Ala Arg Ala Thr Asn Gln Phe Leu Ala Val Ser Lys Gly Asn Cys 85 90 95Ser Gly Pro Thr Thr Ile Arg Gly Gln Phe Ser Asn Met Ser Leu Ser 100 105110 Leu Leu Asp Leu Tyr Leu Ser Arg Gly Tyr Asn Val Ser Ser Ile Val 115120 125 Thr Met Thr Ser Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys130 135 140 Pro Asn Leu Ser Ser Lys Gly Ser Glu Leu Ser Gln Leu Ser MetHis 145 150 155 160 Arg Val Phe Glu Val Gly Val Ile Arg Asn Pro Gly LeuGly Ala Pro 165 170 175 Val Phe His Met Thr Asn Tyr Phe Glu Gln Ser ValSer Asn Asp Phe 180 185 190 Asn Asn Cys Met Val Ala Leu Gly Glu Leu LysPhe Ala Ala Leu Cys 195 200 205 His Arg Glu Asp Ser Ile Thr Ile Pro TyrGln Gly Ser Gly Lys Gly 210 215 220 Val Ser Phe Gln Leu Val Lys Leu GlyVal Trp Lys Ser Pro Thr Asp 225 230 235 240 Met Gln Ser Trp Val Pro LeuSer Thr Asp Asp Pro Val Ile Asp Arg 245 250 255 Leu Tyr Leu Ser Ser HisArg Gly Val Ile Ala Asp Asn Gln Ala Lys 260 265 270 Trp Ala Val Pro ThrThr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr 275 280 285 Cys Phe Gln GlnAla Cys Gln Gly Lys Ile Gln Ala Leu Cys Glu Asn 290 295 300 Pro Glu TrpAla Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val 305 310 315 320 LeuSer Val Asn Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile Ala 325 330 335Ser Gly Phe Gly Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu Tyr 340 345350 Lys Ser Asn His Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro Met Lys 355360 365 Asn Leu Ala Leu Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe370 375 380 Lys Val Ser Pro Asn Leu Phe Thr Val Pro Ile Lys Glu Ala GlyGlu 385 390 395 400 Asp Cys His Ala Pro Thr Tyr Leu Pro Ala Glu Val AspGly Asp Val 405 410 415 Lys Leu Ser Ser Asn Leu Val Ile Leu Pro Gly GlnAsp Leu Gln Tyr 420 425 430 Val Leu Ala Thr Tyr Asp Thr Ser Arg Val GluHis Ala Val Val Tyr 435 440 445 Tyr Val Tyr Ser Pro Ser Arg Ser Phe SerTyr Phe Tyr Pro Phe Arg 450 455 460 Leu Pro Ile Lys Gly Val Pro Ile GluLeu Gln Val Glu Cys Phe Thr 465 470 475 480 Trp Asp Gln Lys Leu Trp CysArg His Phe Cys Val Leu Ala Asp Ser 485 490 495 Glu Ser Gly Gly His IleThr His Ser Gly Met Val Gly Met Gly Val 500 505 510 Ser Cys Thr Val ThrArg Glu Asp Gly Thr Asn Ser 515 520 12 141 PRT Measles virus Attenuatedmeasles virus NA strain 12 Ala Thr Asn Gln Phe Leu Ala Val Ser Lys GlyAsn Cys Ser Gly Pro 1 5 10 15 Thr Thr Ile Arg Gly Gln Phe Ser Asn MetSer Leu Ser Leu Leu Asp 20 25 30 Leu Tyr Leu Ser Arg Gly Tyr Asn Val SerSer Ile Val Thr Met Thr 35 40 45 Ser Gln Gly Met Tyr Gly Gly Thr Tyr LeuVal Glu Lys Pro Asn Leu 50 55 60 Ser Ser Lys Gly Ser Glu Leu Ser Gln LeuSer Met His Arg Val Phe 65 70 75 80 Glu Val Gly Val Ile Arg Asn Pro GlyLeu Gly Ala Pro Val Phe His 85 90 95 Met Thr Asn Tyr Phe Glu Gln Ser ValSer Asn Asp Phe Asn Asn Cys 100 105 110 Met Val Ala Leu Gly Glu Leu LysPhe Ala Ala Leu Cys His Arg Glu 115 120 125 Asp Ser Ile Thr Ile Pro TyrGln Gly Ser Gly Lys Gly 130 135 140 13 7 PRT Measles virus Attenuatedmeasles virus NA strain 13 Leu Glu Ala Arg Ala Thr Asn 1 5 14 7 PRTMeasles virus Attenuated measles virus NA strain 14 Asn Leu Ser Ser LysGly Ser 1 5 15 6 PRT Measles virus Attenuated measles virus NA strain 15Glu Gln Ser Val Ser Asn 1 5 16 7 PRT Measles virus Attenuated measlesvirus NA strain 16 His Arg Glu Asp Ser Ile Thr 1 5 17 1653 DNA Measlesvirus CDS (1)..(1650) Attenuated measles virus CAM-70 strain 17 atg ggtctc aag gtg aac gtc tct gcc ata ttc atg gca gta ctg tta 48 Met Gly LeuLys Val Asn Val Ser Ala Ile Phe Met Ala Val Leu Leu 1 5 10 15 act ctccaa aca ccc acc ggt caa atc cat tgg ggc aat ctc tct aag 96 Thr Leu GlnThr Pro Thr Gly Gln Ile His Trp Gly Asn Leu Ser Lys 20 25 30 ata ggg gtggta gga ata gga agt gca agc tac aaa gtt atg act cgt 144 Ile Gly Val ValGly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg 35 40 45 tcc agc cat cactca tta gtc ata aaa tta atg ccc aat ata act ctc 192 Ser Ser His His SerLeu Val Ile Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 ctc aat aac tgc acgagg gta gag att gca gaa tac agg aga cta ctg 240 Leu Asn Asn Cys Thr ArgVal Glu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 aga aca gtt ttg gaacca att aga gat gca ctt aat gca atg acc cag 288 Arg Thr Val Leu Glu ProIle Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 aat ata aga ccg gtt cagagt gta gct tca ggt agg aga cac aag aga 336 Asn Ile Arg Pro Val Gln SerVal Ala Ser Gly Arg Arg His Lys Arg 100 105 110 ttt gcg gga gta gtc ctggca ggt gcg gcc cta ggc gtt gcc aca gct 384 Phe Ala Gly Val Val Leu AlaGly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125 gct cag ata aca gcc ggcatt gca ctt cac cag tcc atg ctg aac tct 432 Ala Gln Ile Thr Ala Gly IleAla Leu His Gln Ser Met Leu Asn Ser 130 135 140 caa gcc atc gac aat ctgaga gcg agc ctg gaa act act aat cag gca 480 Gln Ala Ile Asp Asn Leu ArgAla Ser Leu Glu Thr Thr Asn Gln Ala 145 150 155 160 att gag gca atc ggacaa gca ggg cag gag atg ata ttg gct gtt cag 528 Ile Glu Ala Ile Gly GlnAla Gly Gln Glu Met Ile Leu Ala Val Gln 165 170 175 ggt gtc caa gac tacatc aat aat gag ctg ata ccg tct atg aac caa 576 Gly Val Gln Asp Tyr IleAsn Asn Glu Leu Ile Pro Ser Met Asn Gln 180 185 190 cta tct tgt gat ttaatc ggc cag aag ctc ggg ctc aaa ttg ctc aga 624 Leu Ser Cys Asp Leu IleGly Gln Lys Leu Gly Leu Lys Leu Leu Arg 195 200 205 tac tat aca gaa atcctg tcg tta ttt ggc ccc agc tta cgg gac ccc 672 Tyr Tyr Thr Glu Ile LeuSer Leu Phe Gly Pro Ser Leu Arg Asp Pro 210 215 220 ata tct gcg gag atatct atc cag gct ttg agc tat gcg ctt gga gga 720 Ile Ser Ala Glu Ile SerIle Gln Ala Leu Ser Tyr Ala Leu Gly Gly 225 230 235 240 gac atc aat aaggtg tta gaa aag ctc gga tac agt gga ggt gat tta 768 Asp Ile Asn Lys ValLeu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu 245 250 255 ctg ggc atc ttagag agc aga gga ata aag gcc cgg ata act cac gtc 816 Leu Gly Ile Leu GluSer Arg Gly Ile Lys Ala Arg Ile Thr His Val 260 265 270 gac aca gag tcctac ttc att gtc ctc agt ata gcc tat ccg acg ctg 864 Asp Thr Glu Ser TyrPhe Ile Val Leu Ser Ile Ala Tyr Pro Thr Leu 275 280 285 tcc gag att aagggg gtg att gtc cac cgg cta gag ggg gtc tcg tac 912 Ser Glu Ile Lys GlyVal Ile Val His Arg Leu Glu Gly Val Ser Tyr 290 295 300 aac ata ggc tctcaa gag tgg tat acc act gtg ccc aag tat gtt gca 960 Asn Ile Gly Ser GlnGlu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala 305 310 315 320 acc caa gggtac ctt atc tcg aat ttt gat gag tca tcg tgt act ttc 1008 Thr Gln Gly TyrLeu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 325 330 335 atg cca gagggg act gtg tgc agc caa aat gcc ttg tac ccg atg agt 1056 Met Pro Glu GlyThr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345 350 cct ctg ctccaa gaa tgc ctc cgg ggg ttc acc aag tcc tgt gct cgt 1104 Pro Leu Leu GlnGlu Cys Leu Arg Gly Phe Thr Lys Ser Cys Ala Arg 355 360 365 aca ctc gtatcc ggg tct ttt ggg aac cgg ttc att tta tca caa ggg 1152 Thr Leu Val SerGly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375 380 aac cta atagcc aat tgt gca tca atc ctt tgc aag tgt cac aca aca 1200 Asn Leu Ile AlaAsn Cys Ala Ser Ile Leu Cys Lys Cys His Thr Thr 385 390 395 400 gga acgatc att aat caa gac cct gac aag atc cta aca tac att gct 1248 Gly Thr IleIle Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr Ile Ala 405 410 415 gac gatcac tgc ccg gta gtc gag gtg aac ggc gtg acc atc caa gtc 1296 Asp Asp HisCys Pro Val Val Glu Val Asn Gly Val Thr Ile Gln Val 420 425 430 ggg agcagg agg tat cca gac gct gtg tac ttg cac aga att gac ctc 1344 Gly Ser ArgArg Tyr Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 435 440 445 ggt cctccc ata tca ttg gag agg ttg gac gta ggg aca aat ctg ggg 1392 Gly Pro ProIle Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 450 455 460 aat gcaatt gct aag ttg gag gat gcc aag gaa ttg ttg gag tca tcg 1440 Asn Ala IleAla Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Ser Ser 465 470 475 480 gaccag ata ttg agg agt atg aaa ggt tta tcg agc act agc ata gtc 1488 Asp GlnIle Leu Arg Ser Met Lys Gly Leu Ser Ser Thr Ser Ile Val 485 490 495 tacatc ctg att gca gtg tgt ctt gga ggg ttg ata ggg atc ccc gct 1536 Tyr IleLeu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile Pro Ala 500 505 510 ttaata tgt tgc tgc agg ggg cgt tgt aac aaa aag gga gaa caa gtt 1584 Leu IleCys Cys Cys Arg Gly Arg Cys Asn Lys Lys Gly Glu Gln Val 515 520 525 ggtatg tca aga cca ggc cta aag cct gat ctt acg gga aca tca aaa 1632 Gly MetSer Arg Pro Gly Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys 530 535 540 tcctat gta agg tcg ctc tga 1653 Ser Tyr Val Arg Ser Leu 545 550 18 550 PRTMeasles virus any n or Xaa = Unknown 18 Met Gly Leu Lys Val Asn Val SerAla Ile Phe Met Ala Val Leu Leu 1 5 10 15 Thr Leu Gln Thr Pro Thr GlyGln Ile His Trp Gly Asn Leu Ser Lys 20 25 30 Ile Gly Val Val Gly Ile GlySer Ala Ser Tyr Lys Val Met Thr Arg 35 40 45 Ser Ser His His Ser Leu ValIle Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 Leu Asn Asn Cys Thr Arg ValGlu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 Arg Thr Val Leu Glu ProIle Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 Asn Ile Arg Pro Val GlnSer Val Ala Ser Gly Arg Arg His Lys Arg 100 105 110 Phe Ala Gly Val ValLeu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125 Ala Gln Ile ThrAla Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser 130 135 140 Gln Ala IleAsp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala 145 150 155 160 IleGlu Ala Ile Gly Gln Ala Gly Gln Glu Met Ile Leu Ala Val Gln 165 170 175Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser Met Asn Gln 180 185190 Leu Ser Cys Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg 195200 205 Tyr Tyr Thr Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro210 215 220 Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu GlyGly 225 230 235 240 Asp Ile Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser GlyGly Asp Leu 245 250 255 Leu Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala ArgIle Thr His Val 260 265 270 Asp Thr Glu Ser Tyr Phe Ile Val Leu Ser IleAla Tyr Pro Thr Leu 275 280 285 Ser Glu Ile Lys Gly Val Ile Val His ArgLeu Glu Gly Val Ser Tyr 290 295 300 Asn Ile Gly Ser Gln Glu Trp Tyr ThrThr Val Pro Lys Tyr Val Ala 305 310 315 320 Thr Gln Gly Tyr Leu Ile SerAsn Phe Asp Glu Ser Ser Cys Thr Phe 325 330 335 Met Pro Glu Gly Thr ValCys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345 350 Pro Leu Leu Gln GluCys Leu Arg Gly Phe Thr Lys Ser Cys Ala Arg 355 360 365 Thr Leu Val SerGly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375 380 Asn Leu IleAla Asn Cys Ala Ser Ile Leu Cys Lys Cys His Thr Thr 385 390 395 400 GlyThr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr Ile Ala 405 410 415Asp Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr Ile Gln Val 420 425430 Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 435440 445 Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly450 455 460 Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu SerSer 465 470 475 480 Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser ThrSer Ile Val 485 490 495 Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu IleGly Ile Pro Ala 500 505 510 Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn LysLys Gly Glu Gln Val 515 520 525 Gly Met Ser Arg Pro Gly Leu Lys Pro AspLeu Thr Gly Thr Ser Lys 530 535 540 Ser Tyr Val Arg Ser Leu 545 550 191653 DNA Measles virus CDS (1)..(1650) Attenuated measles virus NAstrain 19 atg ggt ctc aag gtg aac gtc tct gcc ata ctc atg gca gta ctgtta 48 Met Gly Leu Lys Val Asn Val Ser Ala Ile Leu Met Ala Val Leu Leu 15 10 15 act ctc caa aca ccc acc ggt caa atc cat tgg ggc aat ctc tct aag96 Thr Leu Gln Thr Pro Thr Gly Gln Ile His Trp Gly Asn Leu Ser Lys 20 2530 ata ggg gtg gta ggg ata gga agt gca agc tac aaa gtt atg act cgt 144Ile Gly Val Val Gly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg 35 40 45tcc agc cat caa tca ttg gtc ata aaa tta atg ccc aat ata act ctc 192 SerSer His Gln Ser Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 ctcaat aac tgc acg agg gta gag att gca gaa tac agg aga cta ctg 240 Leu AsnAsn Cys Thr Arg Val Glu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 agaaca gtt ttg gaa cca att aga gat gca ctt aat gca atg acc cag 288 Arg ThrVal Leu Glu Pro Ile Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 aat ataaga ccg gtt cag agt gta gcc tca agt agg aga cac aag aga 336 Asn Ile ArgPro Val Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg 100 105 110 ttt gcggga gtt gtc ctg gca ggt gcg gcc cta ggc gtt gcc aca gct 384 Phe Ala GlyVal Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125 gct cagata aca gcc ggc att gca ctt cac cag tcc atg ctg aac tct 432 Ala Gln IleThr Ala Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser 130 135 140 caa gccatc gac aat ctg aga gca agc ctg gaa act act aat cag gcg 480 Gln Ala IleAsp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala 145 150 155 160 attgag gca atc aga caa gca ggg cag gag atg ata ttg gct gtt cag 528 Ile GluAla Ile Arg Gln Ala Gly Gln Glu Met Ile Leu Ala Val Gln 165 170 175 ggtgtc caa gac tac atc aat aat gag ctg ata ccg tct atg aac caa 576 Gly ValGln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser Met Asn Gln 180 185 190 ctatct tgt gat tta atc ggc cag aag cta ggg ctc aaa ttg ctc aga 624 Leu SerCys Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg 195 200 205 tactat aca gaa atc ctg tca tta ttt ggc ccc agc cta cgg gac ccc 672 Tyr TyrThr Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro 210 215 220 atatct gcg gag ata tcc atc cag gct ttg agc tat gcg ctt gga gga 720 Ile SerAla Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly 225 230 235 240gat atc aat aag gtg tta gaa aag ctc gga tac agt gga ggt gat tta 768 AspIle Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu 245 250 255ctg ggc atc tta gag agc aga gga ata aag gcc cgg ata act cac gtc 816 LeuGly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val 260 265 270gac aca gag tcc tac ttc att gta ctc agt ata gcc tat ccg acg ctg 864 AspThr Glu Ser Tyr Phe Ile Val Leu Ser Ile Ala Tyr Pro Thr Leu 275 280 285tcc gag att aag ggg gtg att gtc cac cgg cta gag ggg gtc tcg tac 912 SerGlu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Ser Tyr 290 295 300aat ata ggc tct caa gag tgg tat acc act gtg ccc aag tat gtt gca 960 AsnIle Gly Ser Gln Glu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala 305 310 315320 acc cag ggg tac ctt atc tcg aat ttt gat gag tca tcg tgt act ttc 1008Thr Gln Gly Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 325 330335 atg cca gag ggg act gtg tgc agc caa aat gcc ttg tac ccg atg agt 1056Met Pro Glu Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345350 cct ctg ctc caa gaa tgc ctc cgg ggg tcc acc aag tcc tgt gct cgt 1104Pro Leu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser Cys Ala Arg 355 360365 aca ctc gta tcc ggg tct ttt ggg aac cgg ttc att tta tca caa ggg 1152Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375380 aac cta ata gcc aat tgt gca tca atc ctc tgc aag tgt tac aca aca 1200Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr 385 390395 400 gga acg atc att aat caa gac cct gac aag atc cta aca tac att gct1248 Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr Ile Ala 405410 415 gcc gat cac tgc ccg gtg gtc gag gtg aac ggt gtg acc atc cag gtc1296 Ala Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr Ile Gln Val 420425 430 ggg agc agg agg tat ccg gac gca gtg tac ttg cac aga att gac ctc1344 Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 435440 445 ggt cct ccc ata tca ttg gag agg ttg gac gtg ggg acg aat ctg ggg1392 Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 450455 460 aat gca att gct aag ttg gag gat gcc aaa gaa ttg ttg gag tca tcg1440 Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Ser Ser 465470 475 480 gac cag ata ttg agg agt atg aaa ggt tta tcg agc act agc atagtt 1488 Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser Thr Ser Ile Val485 490 495 tac atc ctg att gca gtg tgt ctt ggg ggg ttg ata ggg atc cccgct 1536 Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile Pro Ala500 505 510 tta ata tgt tgc tgc agg ggg cgt tgt aac aga aag gga gag caagtt 1584 Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn Arg Lys Gly Glu Gln Val515 520 525 ggt atg tca aga cca ggc cta aag cct gat ctt aca ggg aca tcaaaa 1632 Gly Met Ser Arg Pro Gly Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys530 535 540 tcc tat gta agg tcg ctc tga 1653 Ser Tyr Val Arg Ser Leu 545550 20 550 PRT Measles virus any n or Xaa = Unknown 20 Met Gly Leu LysVal Asn Val Ser Ala Ile Leu Met Ala Val Leu Leu 1 5 10 15 Thr Leu GlnThr Pro Thr Gly Gln Ile His Trp Gly Asn Leu Ser Lys 20 25 30 Ile Gly ValVal Gly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg 35 40 45 Ser Ser HisGln Ser Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 Leu Asn AsnCys Thr Arg Val Glu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 Arg ThrVal Leu Glu Pro Ile Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 Asn IleArg Pro Val Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg 100 105 110 PheAla Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser 130 135140 Gln Ala Ile Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala 145150 155 160 Ile Glu Ala Ile Arg Gln Ala Gly Gln Glu Met Ile Leu Ala ValGln 165 170 175 Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser MetAsn Gln 180 185 190 Leu Ser Cys Asp Leu Ile Gly Gln Lys Leu Gly Leu LysLeu Leu Arg 195 200 205 Tyr Tyr Thr Glu Ile Leu Ser Leu Phe Gly Pro SerLeu Arg Asp Pro 210 215 220 Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu SerTyr Ala Leu Gly Gly 225 230 235 240 Asp Ile Asn Lys Val Leu Glu Lys LeuGly Tyr Ser Gly Gly Asp Leu 245 250 255 Leu Gly Ile Leu Glu Ser Arg GlyIle Lys Ala Arg Ile Thr His Val 260 265 270 Asp Thr Glu Ser Tyr Phe IleVal Leu Ser Ile Ala Tyr Pro Thr Leu 275 280 285 Ser Glu Ile Lys Gly ValIle Val His Arg Leu Glu Gly Val Ser Tyr 290 295 300 Asn Ile Gly Ser GlnGlu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala 305 310 315 320 Thr Gln GlyTyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 325 330 335 Met ProGlu Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345 350 ProLeu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser Cys Ala Arg 355 360 365Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375380 Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr 385390 395 400 Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr IleAla 405 410 415 Ala Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr IleGln Val 420 425 430 Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His ArgIle Asp Leu 435 440 445 Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val GlyThr Asn Leu Gly 450 455 460 Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys GluLeu Leu Glu Ser Ser 465 470 475 480 Asp Gln Ile Leu Arg Ser Met Lys GlyLeu Ser Ser Thr Ser Ile Val 485 490 495 Tyr Ile Leu Ile Ala Val Cys LeuGly Gly Leu Ile Gly Ile Pro Ala 500 505 510 Leu Ile Cys Cys Cys Arg GlyArg Cys Asn Arg Lys Gly Glu Gln Val 515 520 525 Gly Met Ser Arg Pro GlyLeu Lys Pro Asp Leu Thr Gly Thr Ser Lys 530 535 540 Ser Tyr Val Arg SerLeu 545 550 21 23 DNA Artificial Sequence Description of ArtificialSequenceprimer F28 21 agaatcaaga ctcatccaat gtc 23 22 23 DNA ArtificialSequence Description of Artificial Sequenceprimer CF7 22 ttgagagttcagcatggact ggt 23 23 23 DNA Artificial Sequence Description ofArtificial Sequenceprimer CF3 23 acaatgaagt aggactctgt gtc 23 24 23 DNAArtificial Sequence Description of Artificial Sequenceprimer F3 24ggaacctaat agccaattgt gca 23 25 23 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer CF2 25 cgaggtcaat tctgtgcaag tac 23 26 23DNA Artificial Sequence Description of Artificial Sequenceprimer F4 26aaagggagaa caagttggta tgt 23 27 23 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer CF1 27 gatattgttc ggccagaggg aag 23 28 23DNA Artificial Sequence Description of Artificial Sequenceprimer MP5 28atgtcaccac aacgagaccg gat 23 29 23 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer MP4 29 gagattcact gacctagtga aat 23 30 23DNA Artificial Sequence Description of Artificial Sequenceprimer MP2 30tcgctgtccc tgttagactt gta 23 31 23 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer H8 31 gagcaaccag tcagtaatga tct 23 32 23DNA Artificial Sequence Description of Artificial Sequenceprimer MP3 32atgcctgatg tctgggtgac atc 23

What is claimed is:
 1. A measles virus mutant antigen consisting of ameasles virus mutant H protein antigen, wherein said measles virusmutant H protein antigen is at least one member selected from the groupconsisting of the following amino acid sequences (a) to (c) (a) an aminoacid sequence of SEQ ID NO: 10; (b) an amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 11; and (c) an amino acid sequence of SEQ ID NO: 4 orSEQ ID NO:
 12. 2. A recombinant measles virus mutant antigen obtained byreplacing a part of the H protein of CAM-70 strain shown in SEQ ID NO: 2by a corresponding part of the H protein of NA strain shown in SEQ IDNO:
 10. 3. The recombinant measles virus antigen according to claim 2,wherein the 176th to 316th amino acids of SEQ ID NO: 2 are replaced bythe amino acid sequence of SEQ ID NO: 12.